CN104144931B

CN104144931B - Pyrimido [4,5 b] indole derivatives and its application in the amplification of candidate stem cell

Info

Publication number: CN104144931B
Application number: CN201380007006.5A
Authority: CN
Inventors: 居伊·索瓦若; 伊夫·加鲁; 瑞金·鲁埃尔; 斯特凡娜·金格拉斯; 伊曼·法里斯
Original assignee: Universite de Montreal
Current assignee: Universite de Montreal
Priority date: 2012-01-27
Filing date: 2013-01-25
Publication date: 2018-04-10
Anticipated expiration: 2033-01-25
Also published as: CA2862140A1; JP6250556B2; HRP20191189T1; BR112014018524A2; BR112014018524B1; CA2862140C; CN104144931A; LT2807165T; BR112014018524A8; AU2013212457B2; RS59002B1; HUE044070T4; TR201909582T4; PL2807165T3; PT2807165T; KR102098122B1; ES2733946T3; SG11201404241SA; EP2807165A4; KR20140121454A

Abstract

Provide pyrimido [4,5 b] indole derivatives.These compounds can be used for amplifying candidate stem cell group, particularly mankind hemopoietic stem cell group.The compound can also be used in the drug therapy for the disease for being related to candidate stem cell.

Description

Pyrimido [4,5-b] indole derivatives and its in the amplification of candidate stem cell Using

The reference of related application

The benefit of priority for the U.S. Provisional Application No. 61/591,521 submitted this application claims on January 27th, 2012.Will The entire disclosure of which is hereby incorporated by for reference.

Technical field

The present invention relates to pyrimido [4,5-b] indole derivatives.Also, the present invention relates to pyrimido [4,5-b] indoles to spread out Biology is used for the application of amplifying candidate stem cell.In addition, the present invention relates to the treatment of the disease comprising candidate stem cell.

Background technology

The main source of candidate stem cell (HSC) is marrow and Cord blood (UCB).It is (autologous that HSC is used for transplanted sites Or allogeneic), it forms one of maximally effective therapeutic strategy of healing for realizing patient, and the patient is with pernicious Blood disease, marrow failure anaemia (bone marrow failure conditions), various global are concerned about congenital Disease (for example, sickle cell anemia and thalassemia) and autoimmune disease such as lupus.However, due to can not be abundant Ground expands these cells in vitro, makes program safety and successfully, this help or life-improved treatment chance is not useable for In thousands of people all over the world.More specifically, because without discovery HLA (HLA) identical donations Person, for every 3 patients, a chance that transplanting will be abandoned.The patient of other part cannot simply be transplanted, because Grafting (it is, Cord blood or autologous) use for being available for successfully transplanting for HSC very little.The safety of bone-marrow transplantation and have Effect property depends directly on HSC number and the progenitor cells available for grafting.What can be injected is more, and hematological system recovers more fast Speed, and due to granulocyte the infection lacked dangerous window or the dangerous window due to the hematoblastic bleeding lacked It is smaller.The challenge for providing enough HSC further upgrades, wherein such as main inherited blood disorders gene therapy background In (the central genetic reasons of morbidity and mortality all over the world) preferably non-bone marrow suppression conditioning.

In adult, HSC is primarily present in marrow, and must be mobilized, to separate displacement by blood plasma Method is collected, and circulation is advanced into for autologous or allogeneic HSCT (HSCT).The CD34+ of enough numbers The collection of cell, the surrogate markers of (HSC) be it is most important because the success of the Dose Effect Radiation in jury of CD34+ cells and Ratio.Several reports show that the CD34+ cell dosages of higher injection are the independent predictions of the survival of improvement.

Two most generally used mobilization schemes are granulocyte colony stimulating factor (G-CSF) and G-CSF enhanced chemicals Therapy.When being given together with G-CSF, by 2008, U.S.'s food and Drug Administration (FDA) and Her Majesty the Queen in right of Canada as represented by the minister of Healt The Plerixafor of approval in 2011, CXCR4 antagonists improve HSC mobilization.However, because the mobilization of leukaemia, Plerixafor is avoided in the patient with leukaemia.Estimation can not obtain enough numbers using currently used mobilization scheme CD34+ cells/kg influence to reach 15% patient (between disease changing).Often, deposited by normal and cancer stem cell It is marrow and thus, it is possible to which the fact that mobilized, limits applications of the autologous HSCT in malignant hematologic disease.

Allogeneic HSCT with BM or mPBSC is another transplanting alternative.However, about 1 to four/3rd point One of the qualified patient of such transplanting can not find appropriate contributor.For obtaining those transplanted, due to moving Plant versus-host disease, recurrence or graft rejection；With the danger of the immune deficiency of long-term, it is related to there is high-frequency transplanting The death rate.Alternatively, have shown that Cord blood as the effective selection in allogeneic HSCT.However typically, it is single One CB units are used for rapidly for adult patients and effective Radiation in jury provides insufficient HSC.

Support to rebuild the short-term maintenance of the mouse of test measurement or cell factor-mediation of mankind's HSC numbers by mouse or even fit The more sane increase of the progenitor cell of the generally adjoint later type of the increased conditions in vitro of degree.Recently, containing other because Son such as fiber mother cell growth factor (FGF), insulin-like growth factor binding protein, Angiopoietins growth factor and multiple-effect Mouse and mankind HSC more significant increase are described in the culture of growth factor.However, these later reports are so far Proof lonely and that wait is independent.The short-term increase for the HSC that the external cell factor using standard obtains is also inevitable , it is followed by final HSC consumption.

The alternative strategy of mankind HSC amplifications relates to the use of matrix element or solvable form generation part (for example, stimulating Notch, Wnt and Hedgehog path), (PGE2, ROS, p38 and MAPK press down for the targeting of specific intracellular signaling pathway operation Preparation) or specifically their culture of the operation (for example, Hox, Hlf) of transcription factor.For other of HSC in vitro amplification Clinical pathway, which includes, to be utilized：I) purine derivative (StemRegenin 1) (SR1), aromatic hydrocarbon receptor antagonist (Boitano, AE et al."Aryl hydrocarbon receptor antagonists promote the expansion of human hematopoietic stem cells"Science 329:1345-1348.2010)；II) mangosteen alcohol (Garcinol), organize egg White acetyltransferase inhibitors (Nishino, T et al. " Ex vivo expansion of human hematopoietic stem cells by Garcinol,a potentinhibitor of histone acetyltransferase"PLoS ONE 6(9):e24298.2011)；And III) NR-101, non-peptidyl linker small molecule c-MPL activators (Nishino et al. " Ex vivo expansion of human hematopoietic stem cells by a small-molecule agonist of c-MPL"Exp.Hem.2009；37:Incubation 1364-1377).SR1 feature provides low molecule amount (LMW) compound tool There is the proof of the principle for the ability for promoting HSC amplifications.

Clinical research emphasizes not only to need the persistence of the transplanting performed, and it is thin to need to minimize useful grain after transplanting The importance of born of the same parents' horizontal time occurred, the time, in turn, the short-term number multiplied again depending on the cell of injection. Compared with untreated cell, up to the present, clinically without prove in the culture using cell factor the marrow expanded or The useful acceleration of the Radiation in jury of the transplanting of cord blood cell.Examination with the cell using the Notch parts amplification mobilized The earlier results tested show the potential clinical efficacy for any (or even appropriateness) progenitor cells amplification strategy first (Delaney et al."Notch-mediated expansion of human cord blood progenitor cells capable of rapid myeloid reconstitution"Nat.Med.16(2):232-236.2010).However, pass through Need during amplification step ex vivo using the Delta-1 fusion proteins of mobilization and by the record that has to stem cell The shortage (influence seems to be limited to more differentiated progenitor cells) of effect limits this approach.Other approach bags in clinical test Include：I) StemEx, using the common injection of untreated UCB cells, utilize copper chelator tetraethylenepentamine (TEPA) and cell The combination of the UCB cells of factor culture；The result of stage I is shown, compared with previous report, is not improved to neutrophil cell Or time (de Lima M et al. " the Transplantation of ex vivo expanded cord of blood platelet grafting blood cells using the copper chelator tetraethylenepentamine:a phase l/ll clinical trial"Bone Marrow Transplant.2008；41(9):771-778)；With 16-16 dimethyl prostates Plain E2 (PGE2), for improving going back to the nest during UCBT is tested in the stage I.

It is then desired to the new strategy of the amplification for increasing candidate stem cell and progenitor cells.It is used for as is generally known in the art Some pyrimidos [4,5-b] indole derivatives in terms of that；For example, in WO 2003/037898；WO 2004/058764；WO 1998/042708；WO 1997/002266；WO 2000/066585；WO 1993/020078；WO 2006/116733；WO 2008/055233；WO 2010/006032；WO 1995/019970；WO 2005/037825；With public affairs in WO 2009/004329 Open them.However, these documents do not disclose according to the present invention pyrimido [4,5-b] indole derivatives or they in Hematopoietic Stem Application in the amplification of cell and progenitor cells.

Brief description of the drawings

Fig. 1：Compound is not acted on by aromatic hydrocarbons (AhR) approach.Using DMSO, SR1 [AhR antagonists 1000nM], and change Peripheral blood CD34 (+) cell culture for having mobilized 12 hours harvesting and is carried out AhR- response genes by compound 1 [500nM] The real-time quantitative RT-PCR of (CYP1B1 and AhRR).Compound 1 is different from SR1, does not suppress AhR- downstream target genes, shows, it Functional independence in AhR approach.

Fig. 2：The effect of compound 1 is reversible.Detergent compound 1 (being shown with green) or medium (DMSO, with indigo plant Color is shown) peripheral blood CD34 (+) cell of the mobilization of culture 7 days, and it is seeded in again and added with and without compound Add in the fresh culture of thing (respectively, solid line is to dotted line).By 8 days and detection CD34+CD45RA- of cell culture in addition Percentage.When washing compound 1 off, it was observed that the rapid reduction of CD34+CD45RA- percentages, instruction, its effect is reversible 's.

Fig. 3：Flt3, SCF and TPO are that the expansion of stem cells that compound 1 mediates needs.In growth factor (Flt3+SCF + TPO) it is presence or absence of in the case of, by peripheral blood CD34 (+) cell culture for having mobilized 7 days.Lack any growth because The period of the day from 11 p.m. to 1 a.m, CD34+CD45RA- cell counts are relatively low, show, the needs of their pair effects observed.

Fig. 4：A) compound 1 reduces the differentiation for the peripheral blood cells that CD34+ is mobilized.As determined by facs analysis, with The control cell of DMSO processing is compared, and compound 1 can expand CD34+ cell masses.SR1 cooperates with compound 1 keeps what is expanded Cell CD34+ is cultivated, is shown, the suppression using the differentiation of combination is even more notable.B) compound 1 reduces CD34+ Cord bloods The differentiation of cell.C) compound 40 reduces the differentiation of CD34+ cord blood cells.D) compound 40 is held back CD34+ cell differentiations The existing dose-dependent effects of tabulation.

Fig. 5：A) compound 1 expands CD34+ the and CD34+CD45RA- cell masses in the peripheral blood-source mobilized in vitro. Although in the cell that DMSO and compound 1 are handled, total cell count is identical, and CD34+ colonies exceed twice of DMSO. Similarly, the Zu Zhong CD34+CD45RA- colonies that compound 1 is handled exceed to three times in DMSO.Utilize the common point with SR1 Reason strengthens this observation.B) compound 1 strengthened CD34+ the and CD34+CDRA- cells of Cord Blood-Derived in 7- days incubation periods Amplification.C) amplification in vitro table of the compound 1 in 12- days incubation periods to CD34+ the and CD34+CDRA- cells of Cord Blood-Derived Now positive effect.

Fig. 6：, will in the presence of medium (DMSO), SR1, compound 1 and compound 1 and SR1 combination CD34+mPB cell culture ten days.The achievement of the cell of 50,000 and 500,000 processing is transplanted in NSG mouse, and moved After plant after 13 weeks, bone marrow analysis is carried out, to evaluate mankind's hematopoietic transplant survival rate.Give on cell dosage, utilize any The CD34+mPB cell transplantations of the processing of compound 1 are better than what DMSO was handled.It is interesting that hundred of mankind's CD45+ cells in BM Divide than being highest in NSG mouse, compared with single compound, its reception is handled thin using combination (compound 1+SR1) Born of the same parents.

Fig. 7：A) cell that compound 1 expands can rebuild human hematopoietic progenitor cells in NSG mouse.It will utilize the medium (DMSO), the achievement of SR1, compound 1 and combined treatment 5,000CD34+CB cells be transplanted to immunologic inadequacy NSG it is old In mouse.After the transfer after 8 weeks, in addition to DMSO, bone marrow analysis shows mankind's CD45+ graftings in all treatment groups. Show that highest grafting is horizontal using the cell of combination (compound 1+SR1) processing.B) in 12 days external incubation periods, compound 40 prevent to be capable of the loss of the human hematopoietic progenitor cells of grafting NSG mouse marrow.

Fig. 8：As by flow cytometry measure, shown using the primitive cell in the bioreactor of feeding culture process Type (CD34⁺、CD34⁺CD90⁺And CD34⁺CD45RA⁺) amplification.The stream that FB compareed=do not had compound 40 adds culture, Cpd40= Stream with compound 40 adds culture.

The content of the invention

Inventor has discovered that some pyrimidos [4,5-b] indole derivatives.These compounds are dry to expanding hemopoietic thin Born of the same parents group is useful, particularly mankind hemopoietic stem cell group.Chemicals in the treatment of disease of candidate stem cell is related to and Useful.

According on one side, the present invention provides general formula I, II, III, IV, V and VI compound：

Substituent in above-mentioned formula I, II, III, IV, V and VI, i.e. Z, W, L, Li, X, X_i、R¹、R²、R³、R⁴With m and this Text is defined below identical.

According on one side, the invention provides the drug regimen of the compound including formula I, II, III, IV, V and VI Thing.

According on one side, the invention provides formula I, II, III, IV, V and VI compound to do for expanding hemopoietic The application of cell.In embodiments of the present invention, candidate stem cell is human cell.

According on one side, the present invention provides a kind of method for being used to increase candidate stem cell or progenitor cells, methods described It is included in the presence of formula I, II, III, IV, V or VI compound, cultivates initial cell population.In the embodiment party of the present invention In formula, initial cell population is internal, external or in vitro.Equally, in embodiments of the present invention, initial cell population bag Include the CD34+ cells from peripheral blood (mPB), marrow (BM) or Cord blood (UCB) harvest mobilized.In addition, according to this hair In the embodiment of bright method, alternatively, in the presence of formula I, II, III, IV, V and VI compound, together with conduct At least one cell proliferation factor of biology or other small molecule, carries out the culture of initial cell population.

According to one side, the invention provides the cell colony of the method according to the invention amplification, more specifically, utilizing The cell colony expanded according to the compound of the present invention.In embodiments, the invention provides the method according to the invention expansion The candidate stem cell of increasing, more specifically, utilizing the candidate stem cell expanded according to the compound of the present invention.

According on one side, the invention provides hematopoiesis disorder/malignant tumour for the treatment of subject, autoimmune disease And/or the method for hereditary immunodeficiency disease, methods described, which includes giving, needs the subject of such treatment to utilize formula I, II, III, IV, V or VI candidate stem cell of compound amplification or formula I, II, III, IV, V or VI compound.

In embodiments of the present invention, hematopoiesis disorder/malignant tumour, autoimmune disease and/or hereditary immune lacks Falling into disease includes marrow failure anaemia, the congenital disorders of various global care (for example, in sickle cell anemia and ground Extra large anaemia), lupus, acute myelogenous leukemia, acute lymphoblastic leukemia, chronic myelogenous leukemia, chronic leaching Bar chronic myeloid leukemia, bone marrow proliferative diseases, myelodysplastic syndrome, Huppert's disease, non-hodgkin's lymph Knurl, lymphogranulomatosis, alpastic anemia, pure red cell aplasia, hemoglobinuria, Fanconi anemia, Mediterranean are poor Blood, sickle-cell anaemia, Wiskott-Aldrich syndrome, the birth defects of metabolism are (for example, especially Gaucher disease).

According on one side, it is used to increase stem cell or progenitor cells or is done for expanding hemopoietic thin the invention provides one kind The kit of born of the same parents, the kit include formula I, II, III, IV, V or VI compound and operation instructions.The present invention's In embodiment, kit includes at least one cell proliferation factor, and it is biology or other small molecule.

Embodiment

Inventor has discovered that some pyrimidos [4,5-b] indole derivatives.These compounds can be used for expanding hemopoietic Population of stem cells, specifically, mankind hemopoietic stem cell group.Compound can also be used for the treatment for the disease for being related to candidate stem cell.

There is formula I, II, III, IV, V or the VI being illustrated below according to the compound of the present invention.Such compound Salt or prodrug are also in the range of the compound according to the present invention.

In formula I, II, III, IV, V and VI, restriction that substituent is such as summarized below.

Z is：1)-P(O)(OR¹)(OR¹)、2)-C(O)OR¹、3)-C(O)NHR¹、4)-C(O)N(R¹)R¹、5)-C(O)R¹、 6)-CN、7)-SR¹、8)-S(O)₂NH₂、9)-S(O)₂NHR¹、10)-S(O)₂N(R¹)R¹、11)-S(O)R¹、12)-S(O)₂R¹、 13)-L, 14) optionally with 1,2 or 3 R^AOr R¹Substituent substitution-benzyl, 15) alternatively with being connected to L and heteroaryl Any one or both on one or more R^AOr R¹- L- the heteroaryls of substituent substitution, 16) alternatively with being connected to L With heterocyclic group any one or both on one or more R^AOr R¹- L- the heterocyclic radicals, 17) optional of substituent substitution One or more Rs of the ground in any one for being connected to L and heteroaryl or both^AOr R¹- L- the aryl of substituent substitution, 18) alternatively with one or more R^AOr R¹Substituent substitution-heteroaryl or 19) alternatively with one or more R^AOr R¹Substituent substitution-aryl.In this list, if it is not present, each substituent is alternatively coupled to L groups On；Also, work as (R¹) and R¹When being connected on nitrogen-atoms, alternatively, they are combined together with nitrogen-atoms, to form 3 to 7- Yuan of rings, it alternatively includes other one or more hetero atoms selected from N, O and S, alternatively with one or more R¹Or R^A Substitute ring.

W is H, halogen or is connected to the group of the pyrimido indole nucleus of molecule in the heart by N, O, S or C atom.Alternatively, W includes at least one portion, and it is saturation, undersaturated, linear, side chain the and/or ring with 1 to 20 carbon atom Shape alkyl and/or miscellaneous alkyl.Equally, alternatively, the part includes at least one other hetero atom, and it is N, O or S.Such as skill Art personnel should be understood that the W in the chemical constitution of the compound of the present invention can belong to chemical group generally in the art Multiple classifications.

More specifically, W is：1)-H, 2)-halogen, 3)-OR¹、4)-L-OH、5)-L-OR¹、6)-SR¹、7)-CN、8)-P(O) (OR¹)(OR¹)、9)-NHR¹、10)-N(R¹)R¹、11)-L-NH₂、12)-L-NHR¹、13)-L-N(R¹)R¹、14)-L-SR¹、15)- L-S(O)R¹、16)-L-S(O)₂R¹、17)-L-P(O)(OR¹)(OR¹)、18)-C(O)OR¹、19)-C(O)NH₂、20)-C(O) NHR¹、21)-C(O)N(R¹)R¹、22)-NHC(O)R¹、23)-NR¹C(O)R¹、24)-NHC(O)OR¹、25)-NR¹C(O)OR¹、 26)-OC(O)NH₂、27)-OC(O)NHR¹、28)-OC(O)N(R¹)R¹、29)-OC(O)R¹、30)-C(O)R¹、31)-NHC(O) NH₂、32)-NHC(O)NHR¹、33)-NHC(O)N(R¹)R¹、34)-NR¹C(O)NH₂、35)-NR¹C(O)NHR¹、36)-NR¹C(O)N (R¹)R¹、37)-NHS(O)₂R¹、38)-NR¹S(O)₂R¹、39)-S(O)₂NH₂、40)-S(O)₂NHR¹、41)-S(O)₂N(R¹)R¹、 42)-S(O)R¹、43)-S(O)₂R¹、44)-OS(O)₂R¹、45)-S(O)₂OR¹, 46) optionally with 1,2 or 3 R^AOr R¹Substitution Base substitution-benzyl, 47) alternatively be connected to L and heteroaryl any one or both on one or more R^AOr R¹ Substituent substitution-L- heteroaryls, 48) alternatively be connected to L and heterocyclic group any one or both on one or More R^AOr R¹- L- the heterocyclic radicals of substituent substitution, 49) alternatively use in any one for being connected to L and aryl or both One or more R^AOr R¹- L- the aryl of substituent substitution, 50)-L-NR¹(R¹)、51)-L-)₂NR¹、52)-L-(N(R¹)-L )_n-N(R¹)R¹、53)-L-(N(R¹)-L)_n- heteroaryl, alternatively use in any one for being connected to L and heteroaryl or both One or more R^AOr R¹Substituent substitutes heteroaryl, 54)-L- (N (R¹)-L)_n- heterocyclic radical, alternatively with being connected to L With heterocyclic group any one or both on one or more R^AOr R¹Substituent substitutes heterocyclic radical, 55)-L- (N (R¹)-L)_n- aryl, alternatively use one or more R in any one for being connected to L and heteroaryl or both^AOr R¹Take Substitute aryl, 56)-O-L-N (R for base¹)R¹, 57)-O-L- heteroaryls, alternatively with being connected to any of L and heteroaryl One or more R in one or both^AOr R¹Substituent substitutes heteroaryl, the 58)-O-L- heterocyclic radicals, alternatively with company One or more R being connected in any one of L and heterocyclic group or both^AOr R¹Substituent substitute the heterocyclic radical, 59)- O-L- aryl, alternatively use one or more R in any one for being connected to L and aryl or both^AOr R¹Substituent substitutes Aryl, the 60)-O-L)₂-NR¹、61)-O-L-(N(R¹)-L)_n-N(R¹)R¹、62)-O-L-(N(R¹)-L)_n- heteroaryl, it is optional One or more Rs of the ground in any one for being connected to L and heteroaryl or both^AOr R¹The substituent substitution heteroaryl, 63)-O-L-(N(R¹)-L)_n- heterocyclic radical, alternatively be connected to L and heterocyclic group any one or both on one or More R^AOr R¹Substituent substitutes heterocyclic radical, 64)-O-L- (N (R¹)-L)_n- aryl, alternatively with one or more R^A Or R¹Substituent substitutes aryl, the 65)-S-L- heteroaryls, alternatively with one or more R^AOr R¹Described in substituent substitution Heteroaryl, 66)-S-L- heterocyclic radicals, alternatively with one or more R^AOr R¹Substituent substitutes heterocyclic radical, the 67)-S-L- Aryl, alternatively use one or more R in any one for being connected to L and aryl or both^AOr R¹Described in substituent substitution Aryl, 68)-S-L)₂NR¹、69)-S-L-(N(R¹)-L)_n-N(R¹)R¹、70)-S-L-(N(R¹)-L)_n- heteroaryl, is alternatively used One or more R^AOr R¹Substituent substitutes heteroaryl, 71)-S-L- (N (R¹)-L)_n- heterocyclic radical, alternatively with one Or more R^ASubstituent substitutes heterocyclic radical, 72)-S-L- (N (R¹)-L)_n- aryl, alternatively with one or more R^A Substituent substitutes aryl, the 73)-NR¹(R¹)、74)-(N(R¹)-L)_n-N(R¹)R¹、75)-N(R¹)L)₂-NR¹、76)-(N (R¹)-L)_n-N(R¹)R^A、77)-(N(R¹)-L)_n- heteroaryl, alternatively with one or more R^AOr R¹Described in substituent substitution Heteroaryl, 78)-(N (R¹)-L)_n- heterocyclic radical, alternatively with one or more R^AOr R¹The substituent substitution heterocyclic radical, 79)-(N(R¹)-L)_n- aryl, alternatively with one or more R^AOr R¹Substituent substitutes the aryl, 80)-heteroaryl, can One or more R of selection of land^ASubstituent substitutes the heteroaryl or 81)-aryl, alternatively with one or more R^ASubstitution Base substitutes the aryl.In this list, if it is not present, alternatively, each substituent is connected on L groups； Also, as two R¹When being present on identical nitrogen-atoms, then each R¹Substituent is independently selected from described herein after R¹Value List；And n is equal to the integer of any 0,1,2,3,4 or 5；Also, work as (R¹) and R¹It is optional when being connected on nitrogen-atoms Ground, they are combined together with nitrogen-atoms, and to form 3 to 7- yuan of rings, it alternatively includes one or more selected from N, O and S Other individual hetero atoms, alternatively with one or more R¹Or R^ASubstitute ring.

L is：1)-C_1-6Alkyl, 2)-C_2-6Alkenyl, 3)-C_2-6Alkynyl, 4)-C_3-7Cycloalkyl, 5)-C_3-7Cycloalkenyl group, 6) heterocycle Base, 7)-C_1-6Alkyl-C_3-7Cycloalkyl, 8)-C_1-6Alkyl-heterocyclyl groups, 9) aryl or 10) heteroaryl., can in this list One or two R of selection of land^ASubstituent substitutes alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl group, heterocyclic radical, virtue independently of one another Base and heteroaryl groups.

R¹It is：1)-H、2)-C_1-6Alkyl, 3)-C_2-6Alkenyl, 4)-C_2-6Alkynyl, 5)-C_3-7Cycloalkyl, 6)-C_3-7Cycloalkenyl group, 7)-C_1-5Fluoridized, 8)-heterocyclic radical, 9)-aryl, 10)-heteroaryl, 11)-benzyl or 12) 5- [(3aS, 4S, 6aR) -2- Oxo hexahydro -1H- thiophene [3,4-d] imidazol-4 yl] valeryl.In this list, alternatively with 1,2 or 3 R^AOr R¹Take Dai Ji substitutes alkyl, alkenyl, alkynyl, cycloalkenyl group, fluoridized alkyl, heterocyclic radical, aryl, heteroaryl and benzyl independently of one another Group.

R²It is：1)-H、2)-C_1-6Alkyl, 3)-SR¹、4)-C(O)R¹、5)-S(O)R¹、6)-S(O)₂R¹, 7) alternatively with 1, 2 or 3 R^AOr R¹Substituent substitution-benzyl, 8) alternatively be connected to L and heteroaryl any one or both on one Individual or more R^AOr R¹Substituent substitution-L- heteroaryls, 9) alternatively be connected to L and heterocyclic group any one or One or more R on both^AOr R¹- L- the heterocyclic radicals of substituent substitution, 10) alternatively with being connected to any of L and aryl One or more R in one or both^AOr R¹Substituent substitution-L- aryl, 11) alternatively with one or more R^A Or R¹Substituent substitution-heteroaryl or 12) alternatively with one or more R^AOr R¹Substituent substitution-aryl.At this In individual list, if it is not present, alternatively, each substituent is connected on L groups.

R^AIt is：1)-halogen, 2)-CF₃、3)-OH、4)-OR¹、5)-L-OH、6)-L-OR¹、7)-OCF₃、8)-SH、9)-SR¹、 10)-CN、11)-NO₂、12)-NH₂、13)-NHR¹、14)-NR¹R¹、15)-L-NH₂、16)-L-NHR¹、17)-L-NR⁴R¹、18)- L-SR¹、19)-L-S(O)R¹、20)-L-S(O)₂R¹、21)-C(O)OH、22)-C(O)OR¹、23)-C(O)NH₂、24)-C(O) NHR¹、25)-C(O)N(R¹)R¹、26)-NHC(O)R¹、27)-NR¹C(O)R¹、28)-NHC(O)OR¹、29)-NR¹C(O)OR¹、 30)-OC(O)NH₂、31)-OC(O)NHR¹、32)-OC(O)N(R¹)R¹、33)-OC(O)R¹、34)-C(O)R¹、35)-NHC(O) NH₂、36)-NHC(O)NHR¹、37)-NHC(O)N(R¹)R¹、38)-NR¹C(O)NH₂、39)-NR¹C(O)NHR¹、40)-NR¹C(O)N (R¹)R¹、41)-NHS(O)₂R¹、42)-NR¹S(O)₂R¹、43)-S(O)₂NH₂、44)-S(O)₂NHR¹、45)-S(O)₂N(R¹)R¹、 46)-S(O)R¹、47)-S(O)₂R¹、48)-OS(O)₂R¹、49)-S(O)₂OR¹, 50)-benzyl, 51)-N₃Or 52)-C (- N=N-) (CF₃).In the list, alternatively with 1,2 or 3 R^AOr R¹Substituent substituted benzyl.

In embodiments of the present invention, compound has formula IIA, IIB, IIC, IVA or VIA as shown below.This The salt or prodrug of the compound of sample are also in the range of the compound according to the present invention.

In the embodiments of the present invention of the compound according to above-mentioned formula IIA, R¹, W and R²It is each as defined above 's.

In the embodiments of the present invention of the compound according to above-mentioned formula IIB, W and R²It is each as defined above , and Het is alternatively with one or more R such as herein above limited¹Or R^AThe 3 of substitution are to 7- circle heterocycles.

In the embodiments of the present invention of the compound according to above-mentioned formula IIC, W and R²It is each as defined above 's；R⁵And R⁶It is same or different, and is L as defined above independently of one another, or they are combined together with C, With formed alternatively include selected from N, O and S one or more heteroatomic 5 to 7- yuan of rings, and alternatively with one or More R¹Or R^ASubstitute ring.In further embodiment, ring is 5- yuan of rings and hetero atom is nitrogen-atoms.Still entering In the embodiment of one step, ring includes four nitrogen-atoms.Still in further embodiment, R²It is benzyl.

In the embodiments of the present invention of the compound according to above-mentioned formula IVA, W, L, R¹And R²Each as limited above Fixed.Same also, m, Li, R³And R⁴It is each as defined above.

In the embodiments of the present invention of the compound according to above-mentioned formula VIA, Z is CO₂Me or 2- methyl -2H- four Nitrogen azoles -5- bases；R²It is benzyl, 3- thenyls or 3- picolyls；It is NH-L-N (R with W¹)R¹, wherein L is C_2-4Alkyl and R¹It is C_1-4Alkyl or (R¹) and R¹Nitrogen-atoms in connection is combined together, and to form 3 to 7- yuan of rings, it is alternatively included Other one or more hetero atoms selected from N, O and S, alternatively with one or more R¹Or R^ASubstitute ring.

In embodiments of the present invention, compound of the invention be compound 1 described in table 2 herein below to 55.The salt or prodrug of such compound are also in the range of the compound according to the present invention.

In the further embodiment of the present invention, compound of the invention has described in table 2 following here Formula.The salt or prodrug of such compound are also in the range of the compound according to the present invention.

Definition：

Unless otherwise stated, using following definition：

Unless context is clearly pointed out in addition, otherwise singulative "one", " one kind " and "the" include it is corresponding multiple Number reference.

As employed herein, term " comprising " refers to follow the list of the key element of word " comprising " to be necessary or mandatory , still, other element is optional and there may be or can be not present.

As employed herein, term " Consists of " refers to include and is limited to the anything for following phrase " Consists of " Thing.Thus, the key element that phrase " Consists of " shows to list is necessary or enforceable, and shows can not possibly there is other Key element.

As employed herein, term " alkyl " refers to side chain and straight chain full comprising the carbon atom with defined amount And aliphatic alkyl, for example, by C_1-6C in alkyl_1-6Be defined to be included in linear or branch saturation arrangement have 1,2, 3rd, the group of 4,5 or 6 carbon.C as defined above_1-6The example of alkyl include, but not limited to methyl, ethyl, n-propyl, Isopropyl, normal-butyl, the tert-butyl group, isobutyl group, amyl group and hexyl.

As employed herein, term " cycloalkyl " refers to the fat of the wherein monocyclic saturation of the carbon atom with defined amount Race's hydrocarbyl group, for example, by C_3-7C in cycloalkyl₃-C₇It is defined to that there are 3,4,5,6 or 7 included in the arrangement of monocyclic saturation The group of carbon.C as defined above₃-C₇The example of cycloalkyl include, but are not limited to, cyclopropyl, cyclobutyl, cyclopenta, hexamethylene Base and suberyl.

As employed herein, term " alkenyl " refers to unsaturated straight or branched hydrocarbyl group, wherein with defined amount Carbon atom, and wherein at least two carbon atom is bonded to each other by double bond, and there is E or the Z region chemically combination with it. For example, by C₂-C₆C in alkenyl₂-C₆It is defined to be included in straight or branched arrangement and there are 2,3,4,5 or 6 carbon, at least two The group that individual carbon atom is combined together by double bond.C₂-C₆The example of alkenyl include, but are not limited to, ethylidine (vinyl), 1- acrylic, 2- acrylic, 1- cyclobutenyls etc..

As employed herein, term " alkynyl " refers to the carbon atom and wherein at least two carbon wherein with defined amount Atom is bonded the unsaturation being combined, straight-chain hydrocarbons group by three.For example, by C₂-C₄Alkynyl, which is defined to be included in chain, to be had 2nd, 3 or 4 carbon atoms, the group that at least two carbon atoms are combined by three bonds.The example of such alkynyl includes, but It is not limited to, acetenyl, 1- propinyls, 2-propynyl, etc..

As employed herein, term " cycloalkenyl group " refers to the monocyclic representative examples of saturated aliphatic of the wherein carbon atom with defined amount Hydrocarbyl group, for example, by C₃-C₇C in cycloalkenyl group₃-C₇Being defined to be included in has the base of 3,4,5,6 or 7 carbon in monocyclic arrangement Group.The C of above-mentioned restriction₃-C₇The example of cycloalkenyl group include, but are not limited to, cyclopentenyl, cyclohexenyl group etc..

As employed herein, term " halo " or " halogen " refer to fluorine, chlorine, bromine or iodine.

As employed herein, term " haloalkyl " refers to alkyl as defined above, wherein halogen can be passed sequentially through Atom substitutes each hydrogen atom.The example of haloalkyl include, but are not limited to, CH₂F、CHF₂And CH₃。

As employed herein, term " aryl ", combine individually or with another free radical, refer to containing 6 carbon atoms Aromatic ring carbon monocyclic groups, it can be further fused on the 2nd 5- or 6- member carbon ring groups, and it can be aromatic series , it is saturated or unsaturated.The example of aryl include, but are not limited to, phenyl, dihydro indenyl, 1- naphthyls, 2- naphthyls, tetrahydrochysene Naphthyl etc..Aryl can be connected on another group on the appropriate position on cycloalkyl ring or aromatic rings.

As employed herein, term " heteroaryl " refers to the monocyclic or bicyclic system up to 10 atoms, wherein at least one Individual ring is aromatic, and contains 1 to 4 hetero atom being selected from by O, N and S group formed.Can be via ring carbon atom or one Hetero atom connects heteroaryl.The example of heteroaryl include, but are not limited to, thienyl, benzimidazolyl, benzo [b] thienyl, furan Mutter base, benzofuranyl, pyranose, isobenzofuran-base, benzopyranyl, xanthyl, 2H- pyrrole radicals, pyrrole radicals, imidazoles Base, pyrazolyl, pyridine radicals, pyrazinyl, pyrimidine radicals, pyridazinyl, indyl (indolizinyl), isoindolyl, 3H- indyls, Indyl, indazolyl, purine radicals, 4H- quinolyls, isoquinolyl, quinolyl, phthalazinyl, phthalazinyl (napthyridinyl), quinoxalinyl, quinazolyl, cinnolines base (cinnolinyl), pteridyl, isothiazolyl, different benzo two Hydrogen pyranose, chromanyl, isoxazolyls, furan a word used for translation base, indolinyl, iso-dihydro-indole-group, thiazole [4,5-b-]- Pyridine, tetrazole radical, oxadiazolyls (oxadiazolyl), thiadiazolyl group, thienyl and fluorescein (fluorescein) derivative.

As employed herein, term " heterocycle ", " heterocycle " or " heterocyclic radical " refers to containing selected from being made up of O, N and S 1 to 4 heteroatomic 3,4,5,6 or 7 yuan of non-aromatic ring system of group.The example of heterocycle include, but are not limited to, pyrrolidinyl, Tetrahydrofuran base, piperidyl, 3,5- lupetidines base, pyrrolinyl, piperazinyl, imidazolidinyl, morpholinyl, imidazolinyl, Pyrazoles piperidinyl, pyrazolinyl etc., wherein can be on the nitrogen-atoms of ring as described below or on carbon atom with the connection of ring.

As employed herein, term " alternatively being substituted with one or more substituents " or its equivalent terms are " optional Ground is substituted with least one substituent " refer to situation subsequent description event it is possible that or may not occur, and And refer to that description includes the example for the example of event or situation occur and wherein occurring without.This definition refers to from zero to five substitution Base.

As employed herein, term " subject " or " patient " refer to the mankind and non-human mammal for example primate, cat, Dog, pig, ox, sheep, goat, horse, rabbit, rat, mouse etc..

It is incompatible with synthetic method described herein if instead of base their own, then using to making in these methods Appropriate blocking group (PG) the protection substituent of stable reaction conditions.Can be on the appropriate point of the reaction sequence of method Blocking group is removed, to provide desired centre or target compound.The known appropriate blocking group of those skilled in the art With utilize as appropriate blocking group be used for the method for protecting and deprotect different substituents；Can be in T.Greene and P.Wuts,"Protecting Groups in Chemical Synthesis"(4th ed.),John Wiley&Sons,NY (2007) its embodiment is found in, entire contents are hereby incorporated by for reference.Through the example of the blocking group used It include, but are not limited to, Fmoc, Bn, Boc, CBz and COCF₃.In certain embodiments, can specifically select to be described herein as Method in be under the reaction condition that uses reaction substituent.In these cases, reaction condition turns selected substituent Become another substituent, be useful in the intermediate compound in its method being described herein as or target compound in Desired substituent.

As employed herein, term " pharmaceutical salts " refers to bronsted lowry acids and bases bronsted lowry addition salts.

As employed herein, term " medicinal acid addition salt " refers to those of the biological efficiency for retaining free alkali and performance Salt, its be not biologically or be conversely undesirable, and it is by inorganic acid such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphorus Acid etc., and organic acid such as acetic acid, trifluoroacetic acid, propionic acid, glycolic, pyruvic acid, oxalic acid, maleic acid, malonic acid, butanedioic acid, anti- Butene dioic acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethyl sulfonic acid, p-methyl benzenesulfonic acid, salicylic acid Deng formation.

As employed herein, term " acceptable base addition salt " refers to those of the biological efficiency for retaining free acid and performance Salt, its be not biologically or be conversely undesirable.This can be prepared from inorganic base or organic base to the addition of free acid A little salt.Salt derived from inorganic base include, but are not limited to, sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminium salt etc..It is derivative Include, but are not limited to from the salt of organic base, primary, secondary and tertiary amine salt, the substituted ammonium comprising naturally occurring substituted ammonium, Ring-type ammonium and deacidite, such as isopropylamine, trimethylamine, diethylamine, triethylamine, tripropyl amine (TPA), monoethanolamine, 2- dimethyl Ethylaminoethanol, 2- DEAE diethylaminoethanols, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, sea Baqing mycin (hydrabamine), choline, glycine betaine, ethylenediamine, gucosamine, methylglucamine, theobromine, purine, piperazine, Piperidines, N-ethylpiperidine, polyamino resin etc..

According to the present invention compound or their pharmaceutical salts can contain one or more asymmetric centers, chiral axis and Chiral planes, therefore enantiomter, diastereomer and other stereoisomeric forms in any ratio can be produced and can be according to absolute solid It is chemically defined, such as (R)-or (S)-, or as be used for amino acid (D)-or (L)-.The present invention tends to so may comprising all Isomer, and, their racemic and optically pure form.Can using chiral synthon or chiral reagent come Optically active (+) and (-), (R)-and (S)-or (D)-and (L)-isomer are prepared, or utilizes traditional technology, such as Reversed-phase HPLC is split.Racemic mixture can be prepared, and is hereafter divided into individual optical isomer or can be synthesized by chirality Prepare these optics isomers.Enantiomter can be split by method known to those skilled in the art, for example, logical The formation of diastereo-isomerism salt is crossed, then, crystallization, gas-liquid or liquid chromatography, an enantiomter and mapping can be passed through The selective reaction of isomers specific reagent separates the diastereo-isomerism salt.Those skilled in the art it should also be appreciated that Desired enantiomter is transformed into by another chemical entities by isolation technics, then, it is necessary to which additional step forms the phase The enantiomeric form of prestige.Using optically active reagent, matrix, catalyst or solvent by asymmetric syntheses, or pass through One enantiomter is transformed into another synthesis alternatively specific enantiomter by asymmetric transformation.

Can be existed according to some compounds of the present invention as the mixture of epimer.Epimer refers to Being present in the only one of two or more Stereocenters in respective compound has the diastereoisomer of reverse configuration.

Can be existed according to the compound of the present invention with zwitterionic form and the present invention includes the two of these compounds Property ionic species and its mixture.

In addition, be able to can also be existed according to the compound of the present invention in the form of aqueous and be anhydrous.Comprising being described herein as Any formula compound hydrate.In further embodiment, according to the chemical combination of any formula described herein Thing is monohydrate.In embodiments of the present invention, compound described herein include by weight about 10% or less, about 9% or less, about 8% or less, about 7% or less, about 6% or less, about 5% or less, about 4% or less, about 3% or Less, about 2% or less, about 1% or less, about 0.5% or less, about 0.1% or less water.In other embodiment In, compound described herein includes by weight about 0.1% or more, about 0.5% or more, about 1% or more, about 2% Or more, about 3% or more, about 4% or more, about 5% or more or about 6% or more water.

It is probably convenient or desired that compound is prepared, purified, and/or handled in the form of prodrug.Thus, such as herein Use, term " prodrug " is related to the chemical combination for when making its metabolic (for example, in vivo), producing desired reactive compound Thing.Typically, prodrug is inactive, or the activity fewer than desired reactive compound, but can provide favourable processing, to Medicine or metabolic performance.Unless otherwise noted, otherwise the reference to specific compound also includes its prodrug.

As employed herein, term " EC50 " refers to compared with medium culture (DMSO), cause CD34+CD45RA- cells Count 50% increased concentration.

As employed herein, term " candidate stem cell " or " HSC ", which refer to have, allows them to be divided into functional maturation Cell such as granulocyte (for example, promyelocyte, neutrophil leucocyte, acidophil, basophilic granulocyte), red blood cell are (for example, net is knitted Red blood cell, red blood cell), blood platelet (for example, megakeryoblast, produce megacaryocyte blood platelet, blood platelet) and monokaryon it is thin The versatility of born of the same parents' (for example, monocyte, macrophage), and the cell that can be regenerated when maintaining their versatility.

HSC is the part of initial cell population.Alternatively, the organ of the body of the cell from body or containing hematopoietic origin Obtain these cells.Such source includes unassorted marrow, umbilical cord, peripheral blood, liver, thymus gland, lymph and spleen.For Cell with candidate stem cell characteristic, can be enriched with a manner known to specialists skilled in the present field it is all above-mentioned natural or Unassorted blood product.

As employed herein, term " initial cell population " refers to that identification is included from one in multiple sources mentioned above The HSC of individual harvest cell mass, as known in the art.Initial cell population can be rich in CD34+ cells, it is meant that, based on thin Cellular surface mark CD34+ presence selection cell mass.For example, using flow cytometry and the anti-CD34 antibody of fluorescence labeling Detection and counting CD34+ cells.In addition, initial cell population can be used directly for expanding or freezing and store, for it is later when Between the application put.

During hemoposieis, HSC is branched off into ancestral's stage first, is branched off into marrow pedigree and lymph pedigree, then distinguishes Ground is divided into myeloid stem cell (colony forming cell of mixing, CFU-GEMM) and is divided into lymphoid stem cell.Further Ground, myeloid stem cell via it is red be quick-fried formula formed cell (erythroid burst forming cells) (BFU-E) and Erythroid colonies form cell (CFU-E) differentiation erythroblast, are divided into via megakaryocyte colony forming cell (CFU-MEG) solidifying Haemocyte, cell (CFU-GM) differentiation monoblast, neutrophil leucocyte and basophilic are formed via granulocyte-macrophage colony Property granulocyte, and break up eosinoblast via thermophilic eosin colony forming cell (CFU-Eo), while lymphoid stem cell is via T Lymphoid progenitor cell differentiating into T cell and it is divided into B cell via B lymphoid progenitor cells.By in the presence of cytokine profiles, The Performance Testing of clone these myeloid stem cells that they are formed on soft agar, semisolid methyl cellulose culture medium etc. With a variety of HPCs from them.

The present invention is also included according to the present invention and the compound limited, or its salt herein, is used to treat by under preparing Arrange the application in the medicine of the patient of nonrestrictive list of diseases：By the tested of the above-mentioned disease referred to or body immunological diseases Autologous or allogeneic the transplanting or treatment of person (or patient).The example of malignant hematologic disease/disease and congenital disorders can It include, but are not limited to, acute myelogenous leukemia, acute lymphoblastic leukemia, chronic myelogenous leukemia, chronic lymphatic Chronic myeloid leukemia, bone marrow proliferative diseases, myeloproliferative disorder increase and sign, Huppert's disease, non-hodgkin's lymphomas, Lymphogranulomatosis, alpastic anemia, pure red cell aplasia, hemoglobinuria, Fanconi anemia, thalassemia, Sickle-cell anaemia, Wiskott-Aldrich syndrome, inborn errors of metabolism (for example, especially Gaucher disease).Can Have benefited from the example of the amynologic disease of transplanting much and including multiple sclerosis, lupus, some forms or arthritis, severe Comprehensive immune deficiency etc..

Thus, the present invention includes to give utilizes basis by the patient of any above-mentioned disease/malignant tumour referred to The HSC of the compound amplification of the present invention.

In addition, compound and composition can be used for following nonrestrictive site as described：It is autologous or of the same race different The transplanting of body or the treatment by the above-mentioned disease referred to or the subject (or patient) of the disease of autoimmunity.Hematologic The example of disease/disease and congenital disorders can include, but are not limited to, and acute myelogenous leukemia, Acute Lymphoblastic are white It is blood disease, chronic myelogenous leukemia, chronic lymphocytic leukemia, bone marrow proliferative diseases, myelodysplastic syndrome, multiple Property myeloma, non-hodgkin's lymphomas, lymphogranulomatosis, alpastic anemia, pure red cell aplasia, blood red egg Albiduria, Fanconi anemia, thalassemia, sickle-cell anaemia, Wiskott-Aldrich syndrome, congenital generation Thank to defect (for example, especially Gaucher disease).The example of the amynologic disease of transplanting be can benefit from much and comprising multiple hard Change, lupus, some forms or arthritis, severe combined immunodeficiency etc..

What the present invention also included is to utilize the cell mass obtained afterwards according to method of the invention and described herein amplification Body.Candidate stem cell and progenitor cells can be harvested from adult, Cord blood, fetus or embryonic origin.Utilize method of the invention Cell expands the increase that can cause progenitor cells number, for example, it is accelerating to the time of neutrophil cell and blood platelet grafting In be useful.Such method includes：Include HSC starter population using the reagent culture for the number that can increase HSC.Rise Beginning, colony can be rich in cell surface marker interested or its combination (for example, CD34+, CD34+CD45RA+/-).

Method for expanding HSC

Therefore, the present invention relates to the method for amplifying candidate stem cell, including (a) to provide for including candidate stem cell Beginning cell colony and (b) are under conditions of appropriate for amplifying candidate stem cell, initial cell population described in cultured in vitro.

In a specific embodiment, the method for amplifying candidate stem cell, including (a) are provided including making The initial cell population and (b) of hemocytoblast are in the presence of the compound of the present invention or composition, starting described in cultured in vitro Cell colony.

Cell colony is subjected to enrichment or purification step first, negative comprising the cell based on specific cell marking and/ Or positive selection, to provide initial cell population.It is used for the side for separating the initial cell population based on specific cell marking Guttae Phacosylini is also referred to as flow cytometry or mutual with specific cell surface marker with fluorescence activated cell sorts technology (FACS) The antibody of effect or the solid of ligand binding or insoluble matrix.For example, cell and the solid matrix containing antibody can be made (for example, glass bead column, flask, magnetic-particle) contacts, and removes any uncombined cell.Include magnetic or paramagnetic when using Property bead solid matrix when, cell on bead easily can be attached to by magnetic separtor separation.

In one embodiment, the initial cell population is rich in CD34+ cells.For being enriched with CD34+ cells The method of blood cell population include by Miltenyi Biotec (CD34+ is directly separated kit, Miltenyi Biotec, Bergisch, Gladbach, Germany) or pass through Baxter (Isolex 3000) commercialized kit.

The amount of Cord blood from single tire often not enough treats adult or older children.Utilize the chemical combination of the present invention One advantage of the amplification method of thing or composition is that it can make the Hematopoietic Stem for being only from the sufficient amount of a Cord blood unit The production of cell is possibly realized.

Therefore, in one embodiment, initial cell population is from the newborn Cord blood rich in CD34+ cells Cell.In a related embodiment, the initial cell population derives from one or two Cord blood unit.

In another embodiment, initial cell population has been mobilized from the mankind rich in CD34+ cells Peripheral blood cells.In a related embodiment, the initial cell population is from only from the mankind of patient's separation The peripheral blood cells mobilized.

Preferably, the initial cell population can contain at least 50%CD34+ cells, in some embodiments, exceed 90% CD34+ cells.

Final ratio depending on initial cell population, desired final cell number and desired HSC is changed and is used for The condition of culture of the initial cell population of hematopoietic stem cell expansion.

In a specific embodiment, specifically, the starting from the cord blood cell rich in CD34+ cells is utilized Cell colony, condition of culture include being used for HSC amplifications, other cell proliferation factor type cytokines commonly known in the art With the application of growth factor.Such cell factor and growth factor are probably biological molecule or small molecule and they are included, But it is not limited to, it is IL-1, IL-3, IL-6, IL-11, G-CSF, GM-CSF, SCF, FIT3-L, thrombopoietin (TPO), red Erythropoietin and its analog.As employed herein, " analog " is included as naturally occurring form has biology living The cell factor of property and any structural variant of growth factor, including but not limited to, when with naturally occurring form or cell because Sub- receptor stimulating agent in disclosing WO 2007/145227 in patent such as to the agonist antibodies of TPO acceptors (for example, as being described in detail VB22B sc (Fv) 2, etc.) when, the variant of with enhancing or reduction bioactivity.Select cell factor and growth factor group Close, to expand HSC and progenitor cells, while limit the generation of the cell finally broken up.In a specific embodiment, from by The group selection one or more cell factor and growth factor of SCF, FH3-L and TPO composition.

Pass through (Kishimoto, Ann.review of 1mm.23:2005) 1 describes mankind IL6 or leucocyte Jie Element -6, also referred to as B-cell stimulating factor 2 and be commercially available.(Smith,M Aetal.,ACTA Haematologica,105,3:143,2001) mankind SCF or stem cell factor, also referred to as c-kit parts has been described, fertilizer Maxicell growth factor or steel factor (Steel factor) and be commercially available.Flt3-L or FLT-3 parts, Referred to as FL is bonded to the factor on flt3- acceptors.(Hannum C,Nature 368(6472):643-8) have been described It, and be commercially available.(Kaushansky K(2006).N.Engl.J.Med.354(19):2034-45) retouch TPO or thrombopoietin, also referred to as the megakaryocyte growth factor (MGDF) or c-MpI parts have been stated, and has been that in the market can Buy.

Not only by the way that they are added in culture medium, and by fixing them to matrix or supporter for culture Surface on use chemical composition and biotic component mentioned above, specifically, dissolved in suitably by the composition that will be used Solvent, using the solution coating substrate or supporter of synthesis, and then wash excessive composition off.As can using into Divide to be added to and tentatively scribble in the matrix or supporter of material, wherein the material is attached on composition.

HSC amplification can be carried out in the natural medium, semisynthetic medium or synthetic media according to composition, and According to shape, it may be possible to solid medium, semisolid culturemedium or fluid nutrient medium, and for candidate stem cell and/or hematopoiesis Any nutrient medium of progenitor cells culture, the culture medium is augmented using the mixture of the cell proliferation factor of foregoing description. Such culture medium generally comprises sodium, potassium, calcium, magnesium, phosphorus, chlorine, amino acid, vitamin, cell factor, hormone, antibiotic, blood Clearly, aliphatic acid, carbohydrate etc..In culture, such as situation needs, other chemical compositions can be merged solely or in combination Or biotic component.The such composition being merged in the medium be probably hyclone, human serum, horse serum, insulin, turn Ferritin, lactoferrin, cholesterol, monoethanolamine, sodium selenite, thioglycerol, 2 mercapto ethanol, bovine serum albumin(BSA), acetone Sour sodium, polyethylene glycol, various vitamins, various amino acid, agar, agarose, collagen, methylcellulose, various cells The factor, various growth factors etc..Embodiment suitable for the such basal medium for the method for expanding HSC includes, but unlimited In StemSpan^TMSerum-free amplification culture medium (Canada, Vancouver, stem cells technology), StemSpan^TMWhat H3000- was limited Culture medium (Canada, Vancouver, stem cells technology), CellGro^TM, SCGM (Germany, Freiburg, CellGenix), StemPro^TM- 34SFM (Invitrogen), Dahl Burke Improved Eagle Medium (DMEM), Ham's nutritional blends H12 Mixture F12, McCoy's 5A culture mediums, the Iger limit must culture medium (EMEM), aMEM culture mediums (a improvement Igers The limit must culture medium), RPMI1640 culture mediums, Yi Sikaofu improvement Dulbecco's culture mediums (IMDM), StemPro34 (Invitrogen), X-VIVO 10 (Cambrex), X-VIVO 15 (Cambrex) and Stemline II (Sigma- Aldrich)。

In one embodiment, under the concentration expanded suitable for HSC, in the amplification method phase of the initial cell population Between, give compound of the invention or composition.In a specific embodiment, give be included in 1 to 3000nmol it Between or for example 1 to the concentration between 100nmol the compound or composition.

Initial cell population is substantially by thin from one or two Cord blood unit, or from the PB mobilized wherein In one specific embodiment of born of the same parents or the rich cell compositions of the CD34+ of marrow from harvest, cell is set to expand for HSC Grown under conditions of increasing, for example, amplification at double and characteristic cell colony between 2 to 21 days and/or until obtaining instruction. In one specific embodiment, cell isolated growth under conditions of being expanded for HSC is set to be no more than 21 days, 12 days, 10 days Or 7 days.

Then, washable cell colony, with remove the compound of the present invention or composition and/or cell culture it is any its His composition, and be resuspended in appropriate cell suspension cultures base, it is resuspended for short-period used or in storage culture medium for a long time, example Such as, the culture medium suitable for Cord blood.

Alternatively, after tentatively extracellular matrix or cell adhesion molecule is scribbled, zooblast training can be generally used for HSC and/or HPC are cultivated in foster culture vessel, if any lid culture medium, flask, polybag, Teflon^TMBag.For The material of such coating can be collagen l to XIX, fibronectin, vitronectin, LN1 to 12, nitrogen, solid It is raw albumen, thrombospondin, von Willebrand factor, osteopontin, fibrinogen, various elastin laminins, each Kind proteoglycans, various cadherinses, desmoglein adhesion protein (desmocolin), desmoglein, various integrins, E- choosings Select element, palatelet-selectin, L-selectin, immunoglobulin superfamily, artificial basement membrane, poly- D-Lys, poly-L-Lysine, several Ding Zhi, chitosan, Ago-Gel, marine alga acid gel, hydrogel or its fragment.Such coating material can be with people The recombined material of the amino acid sequence of work modification.By using mechanically control culture medium composition, pH etc. and it can obtain highly dense Spend culture bioreactor culture candidate stem cell and/or HPC (Schwartz R M, Proc.Natl.Acad.Sci.U.S.A.,88:6760,1991；Koller M R,Bone Marrow Transplant,21: 653,1998；Koller,M R,Blood,82:378,1993；Astori G,Bone Marrow Transplant,35: 1101,2005)。

The present invention further provides the cell colony of the HSC with amplification, can by or by foregoing description amplification side Method obtains.In a specific embodiment, it is resuspended so in the medicinal culture medium for being suitable for giving mammalian hosts Cell colony, therefore it provides therapeutic combination.

The present invention further provides having for the transplanting of the autologous or Allogeneic stem cell for mammalian subject to expand The HSC of increasing cell colony or its composition.

For example, referring herein to subject be Marrow Donor or have or be in blood cell levels exhaust or limited Danger in individual.Alternatively, subject is in the Marrow Donor before marrow harvest or the bone after marrow harvest Marrow contributor.Alternatively, subject is the recipient of bone-marrow transplantation.Method described herein is with limited bone marrow reserve It is particularly useful in subject, as above the subject of grade or immune depletion treatment or spinal cord transplantation treatment are previously exposed to as changed Treat the subject of (for example, for treating leukaemia or lymthoma).Alternatively, compared with compareing blood cell levels, subject's tool In the blood cell levels being reduced or the danger in the reduced blood cell levels of development.As employed herein, term control blood Cellular level refers to the subject before the event of subject's blood cell levels is changed or in the event is actually lacked Haemocyte average level.The event package for changing the blood cell levels of subject contains, for example, anaemia, wound, chemotherapy, marrow Transplanting and radiotherapy.For example, subject has anaemia or due to for example, wound is lost blood.

In addition to the candidate stem cell and/or HPC expanded by the method for the present invention, graft can be with Be containing cushioning liquid, antibiotic, medicine composition.

For example, before chemotherapy, radiotherapy or bone-marrow transplantation, simultaneously or afterwards, by the HSC colonies of amplification or including The composition of the cell colony of HSC with amplification gives subject.Alternatively, subject consumes, for example, being characterized in that marrow subtracts Less or the related marrow of inborn, hereditary or acquisition the syndrome of marrow that exhausts.Thus, alternatively, subject is to need Want the subject of hemoposieis.Alternatively, subject be Marrow Donor or have or the danger in the marrow exhausted in Subject.

Candidate stem cell processing is useful as the supplementary therapy of chemotherapy or radiotherapy.For example, HSC is focused on outer Separated in all blood with then from by the subject for undergoing chemotherapy, and after the treatment, give back cell.Thus, subject is experience Or the expected depletion of experience immunocyte treatment such as the subject of chemotherapy, radiotherapy or the contributor for serving as bone-marrow transplantation.Marrow It is one of most voluminous tissue and therefore in vivo, is often first by chemotherapeutics and the organ of radiation damage.As a result, changing Haemocyte generation is promptly destroyed during treatment or radiotherapy, and chemotherapy or radiation must be terminated, to allow hematopoietic cell again Haemocyte supply is supplemented before the secondary patient using chemotherapeutic treatment.Therefore, as described herein, alternatively, will be by being described herein as Method manufacture HSC or haemocyte give the such subject for needing extra haemocyte.

With can in vivo, in vitro or the therapeutic agent of in vitro (for example, small molecule, antibody etc.) enhancing HSC propagation and can Selection of land, at least one pharmaceutical excipient or carrier in combination, there is provided expanded by the compound of the invention or composition of foregoing description The HSCs of increasing.The therapeutic agent of HSC proliferation, which can be strengthened, to be meaned：To the agonist antibodies of TPO acceptors (for example, being disclosed in patent The VB22B sc (Fv) 2, etc. that WO 2007/145227 is described in detail)；Cell factor such as SCF, IL-6, Flt-3 part, TPO or TPO Imitate (for example, such as in WO/2007/022269；WO/2007/009120；WO/2004/054515；WO/2003/103686； WO/2002/085343；WO/2002/049413；WO/2001/089457；WO/2001/039773；WO/2001/034585； WO/2001/021180；WO/2001/021180；WO/2001/017349；WO/2000/066112；WO/2000/035446； WO/2000/028987；Described in WO/2008/028645 etc.)；Granulocyte colony stimulating factor (G-CSF)；Grain macrophage Colony stimulating factor (GM-CSF)；Prostaglandin or Prostanoid receptor agonist in patent (for example, such as disclose WO/2008/ It is described in detail in 073748, prostaglandin E2 acceptor -1 (EP-1) activator, prostaglandin E2 acceptor -2 (EP-2) activator, forefront (EP-3) activator of parathyrine E2 acceptors -3 and prostaglandin E2 acceptor -4 (EP-4) activator)；Tetraethylenepentamine (TEPA)； Notch- parts (Delta-1)；And/or WNT activators.In addition, prevented using mescenchymal stem cell (MSCs) culture stem cell Graft versus host disease (GVHD) and expansion of stem cells can be helped.

It is medicinal refer to be not biologically or be conversely undesirable material, it is, can not cause it is undesirable Biological effect or in harmful manner with the other compositions interaction of the pharmaceutical composition that wherein contains in the case of, by material Give subject or cell.Carrier or excipient are selected, to minimize the degraded of active component and minimize subject or cell Unfavorable side effect.

For any traditional mode compositions formulated of method described herein.Administration is via known to technical staff Effective any approach.For example, oral, non-bowel (for example, intravenous injection), by intramuscular injection, pass through intraperitoneal note Penetrate, endermically, in vitro, intranasally or partly give composition.

The preferable method of administration is intravenous injection.The number of the cell of implantation will allow for factor for example sex, the age, The percentage of cell it is expected in body weight, disease or the type of disorder, disorderly stage, cell colony and produces treatment interests needs Cell amount.In a detailed embodiment, composition is given and including at least >=0.3x by intravenous injection 10⁵CD34⁺/ kg or ＞ 2x 10⁶ CD34⁺Cord blood and 2.5 × 10⁵CD34⁺/ kg or more marrow or the peripheral blood mobilized are thin Born of the same parents.In a specific embodiment, the cell of injection is derived from from all thin of the cord blood cell amplification of single tire Born of the same parents.

For example, in the case of the treatment of leukaemia, can be thin by the candidate stem cell of amplification and/or hematopoiesis ancestral by instiling Born of the same parents' injection utilizes the patient of cancer therapy drug, total body radiation or immunosuppressive drug pretreatment, for the elimination of cancer cell or for supplying The growth encourage of body cell grafting.Suitably, disease, pretreatment and the cell transplant method to be treated is selected by director.Can root Judge the such candidate stem cell transplanted and/or HPC in recipient according to the common experiment used in transplantation treatment In grafting, hemoposieis recovery, transplanting side effect and transplanting therapeutic action presence.

As described above, the invention enables amplifying candidate stem cell and/or HPC, and by using amplification HSC safely and is easily performed transplantation treatment and is possibly realized in a short time.

Also provide herein including one or more containers filled with one or more of compositions described herein Kit.Alternatively, such kit includes needs or desired solution and buffer solution.Alternatively, kit is included and passed through Foregoing description method manufacture stem cell amplification colony or containing be used for manufacture HSC amplification colony container or Composition.Specifically, the present invention is provided to expand the kit of in vitro candidate stem cell, it is included in what is limited in the content of the invention The explanation used of compound and such compound in the method that HSC is expanded, and alternatively, one or more of cell amplifications The factor, or the culture medium for cell growth, specifically, the culture medium for HSC as described above growths.Kit can enter One step includes being used to monitor the antibody of the growth of cell, such as anti-CD34, anti-cd 38 and/or anti-CD45RA antibody.In a tool In the embodiment of body, such kit further includes the one kind being selected from by IL6, FLT3-L, SCF and TPO group formed Or more cell proliferation factor.Alternatively, related to such packaging or kit is operation instructions.

Vivo applications：Also the kit of the compound of the invention of effective dose is provided for, to increase subject's HSC, including over a period for one or more dosage of the compound used, wherein chemical combination of the invention in kit The sum of the dosage of thing is equal to the effective amount of the HSC of increase subject enough.It is from about one day to several days or several for a period of time All or several month.Thus, be for a period of time from least about 5,6,7,8,10,12,14,20,21,30 or 60 days or more or one day and Any number of number of days between 180 days.

Biological experiment

Screening experiment：

In order to identify the activator of new generally acknowledged HSC self-renewings, we adapted high flux basis-screening examination Test, to examine the storehouse of the micromolecular compound (5280 low molecular weight compound) on the CD34+ cells that the primary mankind have mobilized.Should This understands that identical approach is applied to the CD34+ cells from various sources known to those skilled in the art, with separation CD34+ cells.It is micro- using anti-APC magnetic using mouse antihuman CD 34+APC (coming from BD monoclonal antibodies) staining mononuclear cells, and then Ball (MicroBeads) (coming from MACS, Miltenyi Biotec) magnetically marks.Retain magnetic terrestrial reference using AutoMACS posts The cell of note.Our research is with utilization interleukin-6, thrombopoietin, Flt-3 parts and stem cell factor The CD34+CD45RA- cells in the peripheral blood-source for the mobilization cultivated in the culture medium of supplement will promote monocyte (MNC) Amplification, with CD34+CD45RA- colonies reduction and HSC consume the fact based on.Therefore, the low of this loss is prevented Compound molecular weight potentially acts as the activator of HSC amplifications.

In 384- hole plates, trained in the 50 μ l culture mediums containing 1 μm of inspection compound or 0.1%DMSO (medium) Support 2000CD34+ cells/wells.On the starting of experiment and after 7- days are incubated, the ratio of CD34+CD45RA- cells is determined. (DMSO) compared with the control, as determined by the proportional increase of CD34+CD45RA- cells, that examines first is different Six of 5280 compounds of Chemical Background promote the amplification of CD34+CD45RA- cells, and the individual enhancing differentiation in 17 (17). In postsearch screening, the amplification that six compounds of CD34+CD45RA- cell colonys promote is analyzed again.These six compounds In four serve as aromatic hydrocarbon receptor (AhR) antagonist, display promotes the mechanism of action of the in vitro amplification of huCD34+ cells (with SR1 It is identical).In 7- days incubation periods, show that remaining two compounds (it is not aromatic hydrocarbon receptor (AhR) antagonist to be defined as) promote The amplification of MNC comprising CD34+ cells.One in those two remaining compounds is accredited as compound 1 (table 2).

Effect of the compound that following Bioexperiment is used to evaluate the present invention to hematopoietic stem cell expansion.Culture medium：In matchmaker It is situated between (DMSO), in the presence of the combination of the compound or compound of positive control (SR1) or the present invention, the training that uses Support culture medium to be made up of nothing-blood serum medium, the culture medium is supplemented using following recombinant cytokine：Interleukin-6, Thrombopoietin, Flt-3 parts and stem cell factor, each is with 100ng/ml ultimate density.Cell culture：Such as Determined by flow cytometry, the CD34+ cell purities initially harvested are more than 90%.CD34+CD45RA- subpopulations reach high In 70% purity.With 5%CO with 40,000 cells/ml inoculating cells and at 37 DEG C₂It is incubated 7 to 12 days.For long-term training Support, 200,000CD34+ cells/ml from the PB mobilized is inoculated with using serum free medium, wherein in 500nM carrier (DMSO), in the presence of the compound of positive control or the present invention, with interleukin-6, thrombopoietin, Flt-3 Part and stem cell factor supplement the culture medium, and each is with 100ng/ml ultimate density.Cultured in vitro 10 days it Afterwards, compound 1 (table 2) promotes amplifications of the MNCs more than 7- times, CD34+ cells to increase more than 5- times of input value (0 day), and almost More than the value determined for medium, 4- times increases.After cultured in vitro 10 days, the cell with (DMSO) culture that utilizes the medium (22.8 ± 0.9%) are compared, and the cell of compound 1- processing retains high CD34 level of amplification (65.8 ± 5.5%).In addition, with Mordanting compares (4.7 ± 0.4%), and the cell of only compound 1- processing retains highest CD34+CD45RA- colonies Express (24.8 ± 0.9%).Compared with medium, the CD34+CD45RA- cultivated using compound 1 number almost increases 3- times, and More than 7- times of the number of input.Finally, with dosage effect form analysis the present invention compound (concentration range be from 1nM to 5000nM), to determine compared with medium condition, the 50% increased number of valid density of CD34+CD45RA- is produced.In table 2 Show result.

Compound 1 acts on (Fig. 1) not over aromatic hydrocarbons (AhR) approach

Next we prove that influence of the compound 1 to original CD34+CD45RA- primitive hematopoietic cells is in culture It is rapid reversible.This influence of best display in fig. 2, wherein in the presence of compound 1, CD34+ is mobilized Peripheral blood cells culture by 7 days.On that time, compound 1 is removed by washing cell.Green dotted line shows, Ratio (the DMSO for proportionally reducing the culture for following control closely of CD34+CD45RA- cells：Solid blue line and dotted line), however, vertical Culture (solid green line) in 2 weeks is seen, the cell supported in the presence of compound 1 retains more original phenotypes.These results are clear Indicate clearly, in 2 days exposed without compound, as seen in being cultivated in control, cell has obtained differentiation marker.Cause And influence of the compound 1 to hominid's cell is rapid reversible.

Compound 1 is not mitogen (mitogen) (Fig. 3)

We display that, in the case where lacking growth factor, compound 1 independently can not trigger cell to breed.In Fig. 3 These results instruction of display, observes similar to the antagonist using aromatic hydrocarbon receptor (SR1), is lacking 3 growths listed In the case of any one of the factor, i.e. Flt3, TPO and SCF, compound 1 are unable to induced cell proliferation.This instruction, is similar to SR1, the mitogenesis that the influence of the keeping in vitro to original HSC phenotypes of compound 1 is not due on this colony influence, and It is due to the influence of the prevention to cell differentiation.

Compound 1 and compound 40 all prevent cell differentiation and act synergistically (Fig. 4) with AhR antagonists

We are also changed using cytological analysis and flow cytometry evaluation one of compound 1 and its effective derivative Influence of the compound 40 to cell differentiation.Two kinds of studies have shown that compound 1 and its derivative compound 40 prevent cell Differentiation.FACS results are shown in Fig. 4.The influence that Fig. 4 A Compounds 1 break up to the peripheral blood cells mobilized.Expanded at 7 days After increasing, peripheral blood that relatively pure (＞ 85%CD34+) has been mobilized exposed to control (DMSO) or compound 1 or SR1 or Compound 1+SR1.As a result strongly indicate that, in control is cultivated, cell promptly discharges CD34 cell surface expressions, however, logical Cross the SR1 for introducing optimal level and compound 1 partly abolishes this influence.Interestingly, SR1 and compound 1 are remaining thin Synergy is played in CD34 expression on cellular surface.For the Cord blood sample in Fig. 4 B and the lifting of compound 40 1 with Fig. 4 C These multiple observations.

Except this point, we show, the influence of compound 1 or compound 40 to the cell with more original phenotypes is most Significantly.For example, CD34+CD45RA- cells (Fig. 4 A to 4C bottom diagram upper left corner a quarter) are utilizing compound 1 or 40 It is more more in SR1 or in cultivating in control than them in the culture of supplement.Observe compound 1 or chemical combination again in these cultures The synergistic effect of thing 40 plus SR1.

Fig. 4 D provide disappearing for CD34 mark of the indication compound 40 on the surface for the colony for preventing hominid HSC from being enriched with The dose-response curve of the efficiency of disalignment.Record the CD34 changed with the different dosage of compound expression.

Compound 1 and 40 expands mankind HSC phenotypes (Fig. 5) in vitro

Fig. 5 shows, not only compound 1 but also compound 40 is expanding mankind's HSC phenotypes in vitro in short-term and long-term cultivation. Fig. 5 A show that in the peripheral blood (mPB) mobilized using CD34+ is started and supports the culture of 12 days, total cell technology exists Increase about 20 times in input.The level of amplification is also identical, either utilizes compound 1, SR1, compound 1+SR1 or control DMSO starts to cultivate.It is most significantly, it was observed that influence of the compound 1 to more CD34+CD45RA cell subpopulations, is changing The colony expands about 10 times and expands about 15 times in the presence of compound 1+SR1 in the presence of compound 1.This observation connects Strongly suggested that with the result presented in Fig. 4, the cell proliferation of compound 1 does not influence, but prevents CD34+CD45RA cells Differentiation, more mature cells be cost cause their pure amplification.Fig. 5 B result instruction, compound 1 will be at seven days 30-40- times of CD34+CD45RA- cord blood cells is caused to expand on cycle, but these cells expand in the presence of DMSO About 15 times (control).Fig. 5 C show similar result, but the time lengthening cultivated takes to 12 days and by compound 40 For compound 1.Again, as indicated in left figure, total cell amplification it is identical, no matter cell exposed to DMSO (control), SR1, Compound 40 or compound 40 plus SR1.It is more impressive to be, at 12 days in the culture supplemented using compound 40 By the more original CD34+CD45RA- cells of 80 times of amplifications, but these cells are expanded in the culture started using SR1 Slightly less than 20- times, show superiority of this compound on aromatic hydrocarbons inhibitor antagonist.

In a word, these results are shown, compound 1 and compound 40 are to CD34⁺More original CD34⁺CD45RA^-The expansion of colony Increasing has main influence, both has from the peripheral blood or the CD34+ of cord blood cell mobilized.

The ex vivo functional of the cell expanded using traditional culture colony forming unit (CFU-C) experimental check.Passing Under conditions of system, it is seeded in by untreated cell or using the cell of the compound incubation of DMSO, positive control or the present invention In methylcellulose medium.As embodiment, compound 1 (table 2, embodiment 1) expands many pluripotency HPCs.Profit The methylcellulose culture of the 1000CD34+mPB cells of 10 days is handled with compound 1 causes multidirectional granulocyte, red blood cell, macrophage Cell and megacaryocyte (GEMM clones) increase and compared with control cell more than input cell 5- times, cause 10 times of increases. This shows that compound 1 described herein also promotes the amplification of pluripotent progenitor cells.

Utilize influence of the NSG mouse models evaluation compound 1 to the mPB HSC of culture.

Next, we evaluate compound 1 and compound 40 to expanding ten to 12 days in vitro and introducing NSG mouse models Cord blood and the peripheral blood mankind HSC influence mobilized.Purposes of these experiments are to confirm, are also for a long time being refilled The influence for the original HSC phenotypes that our compound is shown to Figure 4 and 5 is observed on stem cell/progenitor cells.Fig. 6 is shown The reconstruction of the mouse marrow of 13 weeks pricer class cells after the transfer.For the blood mobilized, 50 are presented in fig. 6, The achievement of 000 and 500,000 cells.As shown herein, as evaluated in NSG mouse models, in amplification human stem cells In, HSC activators SR1 is as one man better than DMSO (control).Again, compound 1 is in these trials as being better than SR1.Such as exist See in vitro culture, compound 1 and SR1 show synergy (Fig. 6) in these trials.

The influence (Fig. 7) of compound 1 and Cord blood (CB) HSC of 40 pairs of cultures using the evaluation of NSG mouse models

Fig. 7 shows the influence to the Cord blood mankind HSC of the culture of interior evaluating in NSG mouse models.In Fig. 7 A As a result indicate, when compared with compareing culture, reconstruction activity of the compound 1 to human cell has obvious effect.Training in short term These experiments are completed in supporting, it is, 7 days.Most significantly, Fig. 7 B indicate, when using 1500CD34+ cells when, with for The 2% of DMSO controls is compared, the considerable effect of average level of the compound 40 with 10% reconstruction.In this experiment It is probably the longer culture due to being used in the experiment that describes in figure 7b that the effect of compound 40, which is more than compound 1 (Fig. 7 A), Period.In ensuing part, the longer cultivation period of utilization is provided and more tested inside determination for (12 to 16 days).

The influence (Fig. 8) of compound 1 and Cord blood (CB) HSC of 40 pairs of cultures using the evaluation of NSG mouse models

Zandstra et al. is demonstrated recently, and the new method for being called stream plus culture optimizes the body for causing mankind HSC to expand Outer condition (U.S. Patent number US7,795,024).We want to confirm, whether compound of the invention is to the stream of these previous optimizations Condition of culture is added to work.For these researchs, we, which follow, utilizes the external training in 40 12 days or 16 days of stream plus culture+compound Support, evaluate the amplification of the candidate stem cell (HSC) in Cord blood-source.Grafting based on human cell to immunodeficient mice is commented Valency HSC numbers.

As Fig. 8 is shown, until at least 16 days, the addition (Cpd40) of compound 40 was to including CD34+CD45RA- cells The amplifications of colony of all inspections main effect is provided.This act in 12-16 days time points be it is most significant, clearly Ground proves stream plus the synergy of culture (FB in Fig. 8) and compound 40.

In addition, the CD34+ cells being enriched with recently and 12 or 16 days cells expanded are transplanted to female NOD/SCID/IL- In invalid (NSG) mouse of 2R γ c-, before transplanting, it is irradiated into 24h in sub- mortally (250 rad).It is quiet by tail Arteries and veins intravenous injection injection cell.It is determined that time point on (3 weeks, 9 weeks and 16 weeks), animal is put to death, and from two shin bones Marrow is harvested with two femurs.Marrow is depleted of into red blood cell and evaluated by flow cytometry, to quantify the grafting of human cell. It is that grafting is positive by cell record if >=0.5% cell is mankind CD45 and human HLA-ABC positive.Evaluating Each time point on, some animals are transported for independent histologic analysis.

As table 1 below is shown, on the 16- time-of-weeks point in evening, the dose response of the grafting of each condition be present.Have Limit dilution analysis is shown, highest HSC amplifications (18.4- times) are produced by the culture in 40 12 days of stream plus culture+compound.This is aobvious Ground is write higher than by amplification (8.1- times) caused by the culture in 12 days of stream plus culture (batch feed) control.Compared with 16 days cultivate, Cultivated using 12 days, two kinds of conditions produce higher amplification.These the results are provided in compound 40 and in stream plus condition of culture In the presence of it active, the clear and definite proof (Fig. 8+table 1) of external pure mankind HSC amplifications.

Table 1：16 weeks limiting dilution grafting Data Summaries

* pure HSC amplification=(#HSC 0 day)/(#HSC 12 days or 16 days)；It is limited in NSG mouse using at 16 weeks Dilution analysis (LDA) measurement.

Synthetic method

The synthetic method being summarized below is related to embodiments of the present invention, and wherein substituent Z is in pyrimido indole nucleus On 7- positions.Such as technical staff it should be understood that, can perform similar synthetic method, there is the change that technical staff is apparent from Change, for embodiments of the invention, wherein substituent Z is on different positions, e.g., for example, on 5,8 or 6- positions, is had Body on 6- positions.

Scheme 1 describes synthesis of the common precursor (1-VI) to the compound of the present invention.In the first step, in the presence of alkali Under, e.g., but it is not limited to, sodium hydride, aryl fluorides 1-I is handled using alkyl cyanoacetates 1-II.Then, in acetic acid, Using reducing agent, e.g., but it is not limited to, the product 1-III of zinc powder processing synthesis, to provide amino indole 1-IV, is utilizing formyl It is transformed into pyrimidine 1-V in the processing of amine and ammonium formate.Utilize reagent such as POCl3 or tribromo oxygen phosphorus processing compound 1- V, to provide reaction intermediate 1-VI, the product is handled using amine 1-VII, to provide the compound 1-VIII of the present invention.

Scheme 1

Scheme 2 describes compound 2-V preparation.With and without alkali (Morsin M.et al.Chemistry-A European Journal, 2009, vol.15, #6, pp.1468-1477) or palladium catalyst in the case of, carry out 4,6- bis- it is chloro- 5- iodine pyrimidines 2-II and corresponding phenol, aniline or benzenethiol 2-I reaction, to give intermediate product 2-III.Utilize Pd (OAc) the intermediate product 2-III of synthesis is transformed into three ring adduct 2-IV (Zhang M.et al.Tetrahedron by 2 Letters,2002,vol.43,p.8235).Finally, according to the embodiment 1 summarized herein below, compound of the invention is obtained 2-V。

Scheme 2

Scheme 3：Using azanol, then compound 4-II (Tully are handled using dimethyl acetamide dimethylacetal W.R.et al.Journal of Medicinal Chemistry,1991,vol.34,p.2060).This produces compound 5-I.

Scheme 3

Scheme 4：Using propionitrile processing intermediate 1B (embodiment 1 herein below) in HCl/ dioxanes, alkali is followed by Processing, to provide methyl 2- ethyls -4- hydroxyl -9H- pyrimidos [4,5-b] indole -7-carboxylic acid's salt (6-I).Then, according to for reality The program of the description of example 1 is applied, obtains compound 6-II.

Scheme 4

Scheme 5：Since the fluoro- 4- nitrobenzoates 7-I of methyl 3-, and the program according to embodiment 1, obtain compound 7-II。

Scheme 5

It is conventional

The HPLC retention times of report are for reversed-phase HPLC (Agilent, 1200 series) solvent using following condition A:MeOH∶H₂O∶TFA(5∶95∶0.05)；Solvent B:MeOH∶H₂O∶TFA(95∶5∶0.05)；Flow：3.0mL/min；2.0 minute Inside gradient 0 to 100%；Post：ZorbaxC18,3.5 microns, 4.6x 30mm；Wavelength 220nm.

On the 6210G1969A LC/MSD TOF spectrometers from Agilent Technologies or from Agilent Technologies Mass spectrum is recorded on the Quadrupole LC/MS Model G6120B of company, it utilizes following condition：Solvent orange 2 A:AcCN∶H₂O∶ HCOOH(5∶95∶0.05)；Solvent B:AcCN∶H₂O∶HCOOH(95∶5∶0.05)；2.0 minutes inside gradients 0 to 100%；Flow： 0.3mL/min；Post：ZorbaxC18,3.5 microns, 2.1x 30mm；Wavelength 220nm.

Experimental arrangement

Embodiment 1

4- ((3- (piperidinyl-1-yl) propyl group) amino)-9H- pyrimidos [4,5-b] indole -7-carboxylic acid's methyl esters

Intermediate 1A

4- (1- cyano group -2- ethyoxyl -2- oxoethyls) -3- nitrobenzene methyls

At 0 DEG C, 2- cyan-acetic esters (10.9mL, 102mmol) are slowly added into DMF In 60% suspension of the sodium hydride (4.10g, 102mmol) in (125mL), to produce grey suspension.By mixture at 0 DEG C Stirring 15 minutes, and add the DMF of the fluoro- 3- nitrobenzoates (10.2g, 51mmol) of methyl 4- (125mL) solution.At 0 DEG C, the peony mixture of synthesis is stirred 30 minutes and in room temperature 3 hours.Utilize 1N HCl (40mL) and ethyl acetate (40mL) diluted reaction mixture.Separated water layer is extracted using ethyl acetate (3 × 50mL).Combination Organic layer and dry, filter and concentration on anhydrous sodium sulfate, to provide residue (26g), by quick-red, orange, green, blue, yellow (ROGBY) by its Purifying (is started with 100% hexane and adds ethyl acetate increment step by step, to including 100% ethyl acetate), to provide 14.9g Title compound.LCMS m/z 291.0(M-H)^-, retention time (on the HPLC of analysis)=1.76 minutes.

Intermediate 1B

3- ethyl -6- methyl 2- amino -1H- indoles -3,6- dicarboxylic esters

In 500mL round-bottomed flasks, in acetic acid (255mL), methyl 4- (1- cyano group -2- ethyoxyl -2- oxygen second is added Base) -3- nitrobenzoyls acid esters (14.9g, 51.0mmol) and zinc powder (16.7g, 255mmol), to produce grey suspension.In nitrogen In atmosphere, at room temperature, the addition of zinc was completed on 35 minutes, it is suitable heat release.Mixture is heated 15 at 100 DEG C Hour.Allow mixture to cool down, filter and utilize ethyl acetate rinse by diatomite.Evaporation provide residue, dichloromethane- Ground in hexane, and after filtration, there is provided 6.3g title compound.LCMS m/z263.2(M+H)⁺, retention time (on analytical HPLC)=1.90 minutes.

Intermediate 1C

4- hydroxyl -9H- pyrimidos [4,5-b] indole -7-carboxylic acid's methyl esters

In 100mL round-bottomed flasks, addition 3- ethyl 6- methyl 2- amino -1H- indoles -3,6- dicarboxylic ester (1.1g, 4.19mmol), ammonium formate (0.53g, 8.39mmol) and formamide (16.7mL, 419mmol), to produce yellow-brown suspension, 165 DEG C are heated to, is heated 12 hours.Mixture is allowed to be cooled to room temperature, and addition water.Filter sediment, the wind of synthesis Do and be dried overnight under high vacuum, to provide 1.1g title compound.LCMS m/z 244.2(M+H)⁺, retention time (on analytical HPLC)=1.51 minutes.

Intermediate 1D

Chloro- 9H- pyrimidos [4,5-b] the indole -7-carboxylic acid's methyl esters of 4-

In 100mL round-bottomed flasks, by 4- hydroxyl -9H- pyrimidos [4,5-b] indole -7-carboxylic acid's methyl esters (1.1g, 4.5mmol) and the mixture of POCl3 (15mL, 161mmol) is heated to 90 DEG C, heats 16 hours, is cooled to room temperature, and Decompression is lower to evaporate.Residue is suspended in dichloromethane (20mL), and is filtered by diatomite.Evaporation provides and is used as orange solids Title compound (360mg).LCMS m/z 262.0(M+H)⁺, retention time (on analytical HPLC)=2.02 minutes.

Embodiment 1

In 2-5mL microwave vials, chloro- 9H- pyrimidos [4, the 5-b] indoles -7- of 4- in methanol (2mL) are added Carboxylate methyl ester (86mg, 0.33mmol), triethylamine (0.09mL, 0.66mmol) and 3- (piperidin-1-yl) propane -1- amine (0.078mL, 0.49mmol), and in microwave reactor, 140 DEG C are heated the mixture to, heat 15 minutes.Allow mixture It is cooled to room temperature and evaporates under reduced pressure.Raw material are dissolved in DMF, and in anti-phase Zorbax SB- Purified on C18 post 21.2x 100mm, and utilize MeOH- water -0.1%TFA elutions.Gradient：Isocratic 20%4 minutes, then, On 15 minutes, to 100%MeOH gradients.Title compound is obtained as trifluoroacetate (according to those skilled in the art The standardization program known prepares corresponding free alkali and HCl salt).LCMS m/z 368.2(M+H)⁺, retention time is (in analysis On HPLC)=1.38 minutes.

Embodiment 14

7- (1- methyl isophthalic acid H- tetrazolium-5- bases)-N- (3- (piperidinyl-1-yl) propyl group)-9H- pyrimidos [4,5-b] indoles-4- Amine and 7- (2- methyl-2H- tetrazolium-5- bases)-N- (3- (piperidinyl-1-yl) propyl group)-9H- pyrimidos [4,5-b] indoles-4- amine

Intermediate 14A

4- ((3- (piperidinyl-1-yl) propyl group) amino)-9H- pyrimidos [4,5-b] indoles-7- nitriles

Since the fluoro- 3- nitrobenzonitriles of 4-, intermediate 2A is prepared according to the program described in embodiment 1.

Intermediate 14B

N- (3- (piperidin-1-yl) propyl group) -7- (2H- tetrazolium -5- bases) -9H- pyrimidos [4,5-b] indoles -4- amine

In 2-5mL microwave vials, the addition 4- ((3- (piperidinyl-1-yl) propyl group) in (trifluoromethyl) benzene (2mL) Amino) -9H- pyrimidos [4,5-b] indoles -7- nitriles (47.5mg, 0.142mmol) and three normal-butyl Azide tin (409 μ l, 1.491mmol), to produce yellow-brown suspension.Vial is placed in microwave, and is heated to 180 DEG C, heats 30 points Clock.Mixture is concentrated to dryness and added MeOH (3mL) then HCl 4M dioxane solutions (1.07mL, 4.26mmol), To obtain yellow solution.Into the solution of synthesis, diethyl ether (3mL) is added.Stirred 16 hours at 20 DEG C.Received on Buchner Collect the solid obtained.Using diethyl ether (3 × 1mL) and hexane (3 × 1mL) washing block, and under high vacuum, at 30 DEG C Drying solid, until constant weight, to provide the title compound as the 56mg of HCl salt.LCMS m/z 378.2(M+H)⁺, retain Time (on the HPLC of analysis)=1.30 minutes.

Embodiment 14

7- (1- methyl isophthalic acid H- tetrazolium -5- bases)-N- (3- (piperidin-1-yl) propyl group) -9H- pyrimidos [4,5-b] indoles -4- Amine and 7- (2- methyl -2H- tetrazolium -5- bases)-N- (3- (piperidin-1-yl) propyl group) -9H- pyrimidos [4,5-b] indoles -4- amine

In 25mL round-bottomed flasks, in tetrahydrofuran (2mL) and methanol (0.5mL), N- (3- (piperidin-1-yl) are added Propyl group) -7- (2H- tetrazolium -5- bases) -9H- pyrimidos [4,5-b] indoles -4- amine hydrochlorates (43mg, 0.10mmol) and N, N- bis- Wopropyl ethyl amine (36 μ l, 0.21mmol), to produce yellow-brown suspension.Then, trimethyl silicone hydride diazomethane is added (Trimethylsilyldiazomethane) 2M hexane solution (260 μ l, 0.52mmol).By the thin of synthesis at 20 DEG C Yellow suspension stir 3 hours, and addition acetic acid (59 μ l, 1.04mmol).Continue stirring 30 minutes and remove under vacuo Solvent, to provide residue, purified by quick-red, orange, green, blue, yellow (ROGBY) and (started with 100% dichloromethane and little by little add dichloromethane Alkane: methanol: the aqueous oxyammonias of 28%wt. (90: 10: 1) increment, with including 100% dichloromethane: methanol: 28%wt. is aqueous Oxyammonia (90: 10: 1)).The first eluted product obtained is 7- (2- methyl -2H- tetrazolium -5- bases)-N- (3- (piperidines -1- Base) propyl group) -9H- pyrimidos [4,5-b] indoles -4- amine (19mg).LCMS m/z 392.2(M+H)⁺, retention time (analyzing On property HPLC)=1.44 minutes.Second eluted product is 7- (1- methyl isophthalic acid H- tetrazolium -5- bases)-N- (3- (piperidin-1-yl) third Base) -9H- pyrimidos [4,5-b] indoles -4- amine (5mg).LCMS m/z 392.2(M+H)⁺, retention time is (in analysis row HPLC On)=1.27 minutes.

Embodiment 15

NaH (3.41g, 85mmol) is added batch-wise to the 2- cyanoacetamides (7.18g, 85mmol) in DMF (53mL) Cold soln in.After room temperature 30 minutes, the fluoro- 3- nitrobenzene methyls of 4- in 15mL DMF are dropwise added The solution of (8.5g, 42.7mmol).After 3 hours, ice, water and 12mLHCl (10%) mixture are added.Filtering synthesis Solid, rinsed using water and be dried overnight under high vacuum, to produce 9.1g 4- (2- amino -1- cyanogen -2- oxygen ethyl) -3- Nitrobenzene methyl：¹H NMR(400MHz,DMSO-d₆)δppm3.93(s,3H)5.78(s,1H)7.77(s,1H)7.91(d, J=7.83Hz, 1H) 8.04 (s, 1H) 8.39 (dd, J=8.02,1.76Hz, 1H) 8.56 (d, J=1.56Hz, 1H).

Ferric chloride hexahydrate (1.540g, 5.70mmol) and zinc (1.242g, 19.00mmol) are added in DMF The solution of coarse cyanogen-amine compound (0.5g, 1.900mmol) of (4.75mL) and the above-mentioned preparation in water (4.75mL) In, to produce yellow suspension.After heat release, 100 DEG C are heated the mixture to, is heated 45 minutes, and it is then slowly cold But to 20 DEG C, and stir 22 hours.Filter solid is crossed, is washed using DMF (3 × 3mL) and dilutes filtrate using water (40mL), simultaneously In 0 DEG C of stirring.Cross filter solid and utilize water (2 × 5mL) washing block.Solid usually contains impurity.Using EtOAc (3 × 50mL) aqueous layer extracted and the organic layer for utilizing water (50mL) and then utilizing bittern (30mL) washing to merge.In anhydrous MgSO₄On Organic layer, filtering and concentration are dried, to produce 287mg as the solid of brown, the solid is handled using acetone (6mL), with production Raw solid suspension, dilutes the suspension using hexane (5mL).Then, collect solid and under high vacuum, done at 40 DEG C It is dry, until constant weight, to produce the intermediate 15B 2- amino -3- carbamyl -1H- Indole-6-carboxylic acids as pale solid Methyl esters (162mg, 36.6% yield)：¹H NMR(400MHz,DMSO-d₆)δppm 3.80(s,3H)6.62(br.s.,2H) 7.04-7.18(m,2H)7.53-7.63(m,2H)7.72(s,1H)10.80(s,1H)；MS m/z 232.2(M+H)⁺；HPLC Ca.96%, RT=1.37 minute.

By intermediate 15B (0.100g, 0.429mmol), 2- (pyridin-3-yl) methyl acetate (0.130g, 0.858mmol) It is placed in micro-wave oven, and heats with the mixture of sodium methoxide 25%wt MeOH (0.196mL) solution in methanol (0.954mL) To 140 DEG C, heat 45 minutes.After cooling, AcOH (0.050mL, 0.879mmol) is added, and by the slurry of synthesis 20 DEG C stirring 1 hour.Filter solid is crossed, is washed using MeOH (3 × 0.5mL), under high vacuum, in 20 DEG C of dryings, until constant weight, To produce the intermediate 15C as brown solid：4- hydroxyls -2- (pyridine -3- methylene) -9H- pyrimidos [4,5-b] indoles - 7- carboxylate methyl esters (82mg, 57.2% yield)：¹H NMR(400MHz,DMSO-d₆)δppm 3.87(s,3H)4.09(s,2H) 7.34-7.40 (m, 1H) 7.79 (dt, J=8.1,1.8Hz, 1H) 7.83 (dd, J=8.2,1.2Hz, 1H) 7.99 (d, J= 0.8Hz, 1H) 8.02 (d, J=8.2Hz, 1H) 8.48 (dd, J=4.9,1.4Hz, 1H), 8.60 (d, J=2.0Hz, 1H) 12.49 (br.s.,2H):MS m/z 335.2(M+H)⁺；The@220nm of HPLC 95.2% and 92.8%@254nm, RT=1.42 minutes.

By 4- hydroxyls -2- (pyridine -3- methylene) -9H- pyrimidos [4,5-b] indole -7-carboxylic acid's methyl esters (0.050g, 0.150mmol) in POCl₃Mixture in (0.948mL, 10.17mmol) is placed in vial, and is added in micro-wave oven Heat heats 15 minutes to 175 DEG C.After cooling, reactant mixture is poured into frozen water (19mL), it is then aqueous by 50% Being slowly added for NaOH (2.7mL) is basified to pH8, and is finally diluted using EtOAc (20mL), crosses filter solid (the of chloride Once harvest) and using EtOAc (20mL) aqueous layer extracteds and in anhydrous MgSO₄The upper dry organic layer merged, filtering and concentration To drying, to provide the other 35mg harvests of desired chloride derivatives.Directly the chloro- 2- of 4- are used in the next step The receipts of the combination separation of (pyridine -3- methylene) -9H- pyrimidos [4,5-b] indole -7-carboxylic acid's methyl esters (53mg, 100 yield) Obtain.

By the chloro- 2- of 4- (pyridine -3- methylene) -9H- pyrimidos [4,5-b] indole -7-carboxylic acid's methyl esters (0.053g, 0.150mmol), 3- (piperidin-1-yl) propane -1- amine (0.072mL, 0.451mmol) and triethylamine (0.063mL, 0.451mmol) mixture in MeOH (2.5mL) is placed in vial, and 140 DEG C are heated in micro-wave oven, is added Heat 15 minutes.Cool down and solvent evaporation after, by fast chromatographic method purify residue, with produce 23mg yellow oils and Solid, utilize CH₃CN (3mL) dilutes and stirred 30 minutes.Cross filter solid and utilize CH₃CN (2 × 0.5mL) is washed, then in height Spend under vacuum, in 30 DEG C of dryings, until constant weight, to provide the compound as the embodiment 15 of Tan solid：4- ((3- (piperazines Pyridine -1- bases) propyl group) amino) -2- (pyridine -3- methylene) -9H- pyrimidos [4,5-b] indole -7-carboxylic acid's methyl esters (11mg, 16% yield)：1H NMR(400MHz,DMSO-d₆)δppm 1.31-1.44(m,2H)1.44-1.56(m,4H)1.71-1.86 (m,2H)2.17-2.47(m,6H)3.56-3.66(m,2H)3.88(s,3H)4.08(s,2H)7.28-7.34(m,1H)7.43 (t, J=5.5Hz, 1H) 7.76 (dt, J=7.8,2.0Hz, 1H) 7.81 (dd, J=8.2,1.4Hz, 1H) 7.99 (d, J= 1.4Hz, 1H) 8.35 (d, J=8.2Hz, 1H) 8.41 (dd, J=4.7,1.6Hz, 1H), 8.59 (d, J=2.0Hz, 1H) 12.08 (s,1H)；MS m/z 459.2(M+H)⁺；HPLC ＞ 99.5%, RT=1.43 minute.

Embodiment 22

By chloro- 9H- pyrimidos [4,5-b] the indole -7-carboxylic acid methyl esters of 2- ((benzyloxy) (phenyl) methyl) -4- (as implemented Prepared by the description of example 5,0.228g, 0.498mmol), 3- (piperidin-1-yl) -1- amine (0.158mL, 0.996mmol) and triethylamine The mixture of (0.173mL, 1.245mmol) in MeOH (3.8mL) is placed in micro-wave oven, and is heated to 140 DEG C, heating 30 Minute.After cooling to room-temperature, mixture is concentrated to dryness and purifies residue by fast chromatographic method, to provide work For light yellow solid 2- ((benzyloxy) (phenyl) methyl) -4- ((3- (piperidin-1-yl) propyl group) amino) -9H- pyrimidos [4, 5-b] indole -7-carboxylic acid's methyl esters (172mg, 61.3% yield)：¹H NMR(400MHz,DMSO-d₆)δppm 1.29-1.42(m, 2H) 1.42-1.56 (m, 4H) 1.72-1.91 (m, 2H) 2.18-2.47 (m, 6H) 3.66 (tt, J=13.2,6.6Hz, 2H) 3.88 (s, 3H) 4.54 (d, J=11.7Hz, 1H) 4.64 (d, J=12.1Hz, 1H) 5.50 (s, 1H) 7.21-7.43 (m, 8H) 7.51 (t, J=5.9Hz, 1H) 7.57 (d, J=7.0Hz, 2H) 7.82 (dd, J=8.2,1.2Hz, 1H) 8.00 (d, J=1.2Hz, 1H) 8.38 (d, J=8.2Hz, 1H) 12.22 (s, 1H)；HRMS m/z 564.2979(M+H)⁺；HPLC 99.6%, RT=2.02 points Clock.

In methyl alcohol, under hydrogen, in Pd-C 10%wt. (50% weight in wet base) (0.159g, 0.075mmol) and ammonium formate In the presence of (0.235g, 3.73mmol), in derivative 2- ((benzyloxy) (phenyl) methyl) -4- ((3- (piperidin-1-yl) third Base) amino) hydrogenolysis that carries out benzyl on -9H- pyrimidos [4,5-b] indole -7-carboxylic acid's methyl esters (0.042g, 0.075mmol) makees With.After 55 DEG C are stirred 26 hours, reactant mixture is filtered over celite, is rinsed using MeOH, and in Rotary Evaporators On be concentrated to dryness, to produce 133mg residue, the RP HPLC by using Zorbax SB-C18 post 21.2x 150mm are pure Change residue, eluted using MeOH-water -0.1%TFA, the embodiment 22 to provide the 21.9mg as the tfa salt of white solid (50% yield)：2- (hydroxyl (phenyl) methyl) -4- ((3- (piperidin-1-yl) propyl group) amino) -9H- pyrimidos [4,5-b] Yin Diindyl -7- carboxylate methyl esters：¹H NMR(400MHz,DMSO-d₆)δppm 1.30-1.43(m,1H)1.52-1.73(m,3H)1.79 (br.d, J=14.5Hz, 2H) 1.96-2.09 (m, 2H) 2.54 (s, 1H) 2.74-2.89 (m, 2H) 3.11 (dt, J=10.4, 5.4Hz, 2H) 3.38 (d, J=12.1Hz, 2H) 3.71-3.77 (m, 2H) 3.88 (s, 3H) 5.63 (s, 1H) 7.19-7.26 (m, 1H) 7.27-7.35 (m, 2H) 7.48-7.56 (m, 2H) 7.62 (t, J=5.9Hz, 1H) 7.85 (dd, J=8.2,1.4Hz, 1H) 8.03 (d, J=1.4Hz, 1H) 8.38 (d, J=8.2Hz, 1H) 8.92 (br.s., 1H) 12.21 (s, 1H)；HRMS m/z 474.2511(M+H)⁺；HPLC ＞ 99%, RT=1.68 minute.

The high iodine alkane reagent (22.67mg, 0.053mmol) of Dai Si-Martin is added to 2- (hydroxyl (phenyl) methyl) -4- ((3- (piperidin-1-yl) propyl group) amino) -9H- pyrimidos [4,5-b] indole -7-carboxylic acid methyl esters (compound of embodiment 22) and TFA (15.7mg, 0.027mmol) in DCM (1000 μ L, 15.54mmol) mixture, to produce orange solution.1h it Afterwards, evaporation solvent, and residue is purified by fast chromatographic method, to provide the embodiment 23 as bright yellow solid：2- benzene Formyl -4- ((3- (piperidin-1-yl) propyl group) amino) -9H- pyrimidos [4,5-b] indole -7-carboxylic acid's methyl esters (10mg, 79% production Amount)：¹H NMR(400MHz,DMSO-d₆)δppm 1.27-1.36(m,2H)1.36-1.48(m,4H)1.74-1.88(m,2H) 2.14-2.44 (m, 6H) 3.52-3.68 (m, 2H) 3.91 (s, 3H) 7.54 (t, J=7.8Hz, 2H) 7.69 (t, J=7.4Hz, 1H) 7.76 (t, J=5.1Hz, 1H) 7.90 (dd, J=8.2,1.4Hz, 1H) 7.94 (d, J=7.0Hz, 2H) 8.10 (d, J= 1.4Hz, 1H) 8.52 (d, J=8.2Hz, 1H) 12.43 (s, 1H)；HRMS m/z 472.2342(M+H)⁺；The@of HPLC 97.1% @254nm, the RT=1.86 minutes of 220nm and 98.9%.

Embodiment 25

In MeOH (4.00mL) and pyridine (0.666mL), hydroxylamine hydrochloride (0.08g, 1.2mmol) is added to 4- ((3- (piperidin-1-yl) propyl group) amino) -2- (3- (2,2,2- trifluoroacetyl groups) benzyl) -9H- pyrimidos [4,5-b] indoles -7- carboxylics In sour methyl esters (compound of embodiment 33) (0.285g, 0.515mmol), to produce yellow solution.Heated at 60 DEG C 5 days it Afterwards, mixture is concentrated to dryness, and residue is dissolved into DCM (75mL) and MeOH (15mL), and utilize saturation NaHCO₃ (20mL) washs the solution.Utilize CH₂Cl₂Water layer is extracted twice by (50mL) and MeOH (10mL) mixture, and anhydrous MgSO₄The upper dry organic layer merged, filters and is concentrated to dryness, to produce the 4- ((3- (piperidines -1- as pale solid Base) propyl group) amino) -2- (3- (2,2,2- tri- fluoro- 1- (isonitroso) ethyl) benzyl) -9H- pyrimidos [4,5-b] indoles -7- Carboxylate methyl ester (293mg, 100% yield)：¹H NMR(400MHz,DMSO-d₆)δppm 1.32-1.43(m,2H)1.44-1.56 (m,4H)1.76-1.87(m,2H)2.23-2.44(m,6H)3.57-3.67(m,2H)3.88(s,3H)4.04-4.12(m,2H) (d, J=7.4Hz, the 1H) 7.81 of 7.25-7.33 (m, 1H) 7.34-7.46 (m, 2H) 7.50 (m, J=7.6,4.1Hz, 1H) 7.55 (dd, J=8.2,1.2Hz, 1H) 7.99 (d, J=1.2Hz, 1H) 8.36 (d, J=8.2Hz, 1H) 12.07 (d, J=3.5Hz, 1H)；MS m/z 569.2(M+H)+；HPLC ＞ 95%, RT=1.88 minute.

By paratoluensulfonyl chloride (0.048g, 0.251mmol) be added in batches 4- ((3- (piperidin-1-yl) propyl group) amino)- 2- (3- (2,2,2- tri- fluoro- 1- (isonitroso) ethyl) benzyl) -9H- pyrimidos [4,5-b] indole -7-carboxylic acid's methyl esters (0.130g, 0.229mmol), 4-dimethylaminopyridine (2.79mg, 0.023mmol) and triethylamine (0.038mL, 0.274mmol) in DCM (10.00mL) cold mixt, to produce white suspension.After room temperature 1 hour, DCM is utilized (10mL) dilutes amber solution and utilizes water washing (3 × 10mL).In anhydrous MgSO₄Upper dry organic layer, filtering and concentration To drying, to produce 4- ((3- (piperidin-1-yl) propyl group) amino) -2- (3- (2,2,2- tri- fluoro- 1- as yellowish-brown foam ((tosyl) imino group) ethyl) benzyl) -9H- pyrimidos [4,5-b] indole -7-carboxylic acid's methyl esters (188mg, 99% yield)： MS m/z 723.2(M+H)+；HPLC ＞ 89%, RT=2.09 minute.

To 4- ((3- (piperidin-1-yl) propyl group) amino) -2- (3- (fluoro- the 1- ((toluene of 2,2,2- tri- for being cooled to -78 DEG C Sulphonyl) imino group) ethyl) benzyl) -9H- pyrimidos [4,5-b] indole -7-carboxylic acid's methyl esters (0.188g, 0.260mmol) DCM In (5.00mL) solution, addition ammonia (1.689mL, 78mmol) and seal pipe, and it is warmed to 20 DEG C.Reactant mixture with when Anaplasia is blue and after 3.5 hours, and it is cooled again to -78 DEG C, and is then slowly warmed it using barrier film+nitrogen outlet To 20 DEG C, to evaporate most ammonia.After 3.5 hours, reactant mixture is filtered on Buchner, to remove major part P-methyl benzenesulfonic acid ammonium salt, washed using DCM (3 × 1.5mL) and solid and concentrate the filtrate to drying, to produce yellow colored foam, Purified by fast chromatographic method, to provide 4- ((3- (piperidin-1-yl) propyl group) amino) -2- (3- as white foam (3- (trifluoromethyl) diazacyclo propane -3- bases benzyl) -9H- pyrimidos [4,5-b] indole -7-carboxylic acid's methyl esters (115mg, 78% yield)：¹H NMR(400MHz,DMSO-d₆) δ ppm 1.31-1.43 (m, 2H) 1.50 (five one group, J=5.3Hz, 4H) 1.80 (dt, J=14.1,7.0Hz, 2H) 2.32 (m, J=6.7,6.7Hz, 6H) 3.58-3.67 (m, 2H) 3.88 (s, 3H) 3.93 (br.d, J=7.4Hz, 1H) 4.05 (br.d, J=8.6Hz, 1H) 4.07 (s, 2H) 7.32-7.43 (m, 3H) 7.47 (m, J= 6.3Hz, 1H) 7.59 (s, 1H) 7.81 (dd, J=8.4,1.2Hz, 1H) 7.99 (d, J=1.2Hz, 1H) 8.35 (d, J= 8.4Hz,1H)12.07(s,1H)；MS m/z 568.2(M+H)⁺；HPLC ＞ 94%, RT=1.72 minute.

Iodine (0.028g, 0.111mmol) is added to 4- ((3- (piperidin-1-yl) propyl group) amino) -2- (3- (3- (trifluoros Methyl) diazacyclo propane -3- bases benzyl) -9H- pyrimidos [4,5-b] indole -7-carboxylic acid's methyl esters (0.060g, 0.106mmol) In mixture of the triethylamine (0.044mL, 0.317mmol) in DCM (2mL), to produce yellow solution.15 minutes it Afterwards, evaporation solvent under reduced pressure, to produce residue, is purified by fast chromatographic method, to provide 95mg yellow colored foam.Will Foam dissolves in DCM (15mL) and utilizes sat.NaHCO₃(10mL) is washed.In anhydrous MgSO₄Upper dry organic layer, filtering and concentration To drying, the compound to provide embodiment 25 as faint yellow solid：4- ((3- (piperidin-1-yl) propyl group) amino) -2- (3- (3- (trifluoromethyl) -3H- diazacyclo propane -3- bases benzyl) -9H- pyrimidos [4,5-b] indole -7-carboxylic acid's methyl esters (50mg, 84% yield)：1H NMR (400MHz, DMSO-d6) δ ppm 1.36 (m, J=5.1Hz, 2H) 1.48 (five one group, J= 5.5Hz, 4H) 1.76 (five one group, J=7.0Hz, 2H) 2.30 (br.t, J=6.5,6.5Hz, 6H) 3.54-3.67 (m, 2H) 3.88 (s, 3H) 4.09 (s, 2H) 7.14 (d, J=7.8Hz, 1H) 7.26 (s, 1H) 7.38-7.47 (m, 2H) 7.52 (d, J= 7.4Hz, 1H) 7.81 (d, J=8.2Hz, 1H) 7.99 (s, 1H) 8.35 (d, J=8.2Hz, 1H) 12.06 (s, 1H)；HRMS m/z 566.2497(M+H)⁺；The@220nm of HPLC 94.5% and 92.9%@254nm, RT=2.05 minutes.

Embodiment 33 and 34

CO is filled with 2- (3- benzyl bromides) -4- ((3- (piperidin-1-yl) propyl group) amino) -9H- pyrimidos [4,5-b] Yin Diindyl -7- carboxylate methyl esters (as prepared for embodiment 15,0.090g, 0.168mmol), triethyl silicane (0.054mL, 0.336mmol) and PdCl₂(dppf) in the solution of (6.14mg, 8.39 μm of ol), and mixture is stayed overnight into heating at 95 DEG C. Coarse mixture is purified by preparation HPLC, to produce the 44mg carbonylation products as solid：1H NMR(400MHz, DMSO-d6)δppm 1.28-1.40(m,1H)1.50-1.72(m,3H)1.73-1.84(m,2H)1.95-2.06(m,2H) 2.74-2.87(m,2H)3.03-3.12(m,2H)3.33-3.41(m,2H)3.88(s,3H)4.20(s,2H)7.46-7.60(m, 2H) 7.73 (d, J=7.83Hz, 1H) 7.79 (d, J=7.43Hz, 1H) 7.84 (dd, J=8.22,1.57Hz, 1H) 7.91 (s, 1H) 8.01 (d, J=1.17Hz, 1H) 8.37 (d, J=8.61Hz, 1H) 8.92 (br.s., 1H) 10.00 (s, 1H) 12.16 (s, 1H)。

Trimethyl (trifluoromethyl) silane (0.7mL, 3.5mmol) is added to be cooled to 0 DEG C 2- (3- formyls benzyl)- 4- ((3- (piperidin-1-yl) propyl group) amino) -9H- pyrimidos [4,5-b] indole -7-carboxylic acid's methyl esters (0.260g, 0.535mmol) In the mixture of cesium fluoride (5.69mg, 0.037mmol).It is stirred at room temperature after 2 days, the addition concentration HCl in 2mL water (0.5mL), and stir 15min.Using ethyl acetate diluted mixture, solid Na is utilized₂CO₃Neutralize, phases were separated and utilizes EA Water layer is extracted 2 times.The organic layer merged using water washing, in anhydrous MgSO₄Upper drying, filtering and removal solvent, to produce Residue, purified by preparation HPLC, to produce the corresponding tfa salts of 126mg：1H NMR(400MHz,DMSO-d6)δppm 1.24-1.43 (m, 1H) 1.49-1.73 (m, 4H) 1.73-1.82 (m, 2H) 1.97-2.07 (m, 2H) 2.80 (q, J= 11.70Hz, 2H) 3.02-3.12 (m, 2H) 3.36-3.42 (m, 2H) 3.88 (s, 3H) 4.10 (s, 2H) 5.12 (q, J= 7.17Hz,1H)6.81(br.s.,1H)7.30-7.35(m,2H)7.36-7.42(m,1H)7.48-7.58(m,2H)7.84(dd, J=8.41,1.37Hz, 1H) 8.01 (s, 1H) 8.37 (d, J=8.61Hz, 1H) 8.95 (br.s., 1H) 12.17 (s, 1H)；MS m/z 554.2(M+H)+；HPLC RT 2.142 minutes.

The high iodine alkane (56.5mg, 0.133mmol) of Dai Si-Martin is added to the 4- ((3- (piperidines -1- in DCM (753 μ L) Base) propyl group) amino) -2- (3- (tri- fluoro- 1- of 2,2,2- ((ethoxy) benzyl) -9H- pyrimidos [4,5-b] indole -7-carboxylic acid's first In the solution of ester (20mg, 0.036mmol), to produce white suspension.After 20 DEG C are stirred 1 hour, pass through flash chromatography Analytic approach purified mixture, to produce the embodiment 33 as yellow solid, 4- ((3- (piperidin-1-yl) propyl group) amino) -2- (3- (2,2,2- trifluoroacetyl groups) benzyl) -9H- pyrimidos [4,5-b] indole -7-carboxylic acid's methyl esters：In DMSO-d6¹H NMR It is consistent with desired product, but because the presence of hydrate forms is more complicated；HRMS m/z554.2384(M+H)⁺；HPLC ＞ 95%, RT=1.76 and 1.87 minutes (ketone+hydrate).

Embodiment 35

Intermediate 35B：N2 isomers precursors

By p-Fluorophenyl cyanide (5g, 41.3mmol), Dibutyltin oxide (2.055g, 8.26mmol) and trimethyl silane Mixture of the azide (8.22mL, 61.9mmol) in toluene (165mL) is heated to 100 DEG C, and stirs 16.5 hours. It is cooled to after room temperature, extracts organic layer using NaOH 1M (83mL), and utilize EtOAc (2 × 85mL) washing water layers.Utilize Water layer is acidified to pH2 by HCl 2M (41.3mL).Aqueous mixture is extracted twice using EtOAc (200mL and then 100mL), And the organic layer merged using bittern (60mL) washing, in anhydrous MgSO₄Upper drying, filter and be concentrated to dryness, to provide Intermediate (5- (4- fluorophenyls) -2H- tetrazoliums, 6.61g, 98% yield) as white solid：¹H NMR(400MHz,DMSO- d₆)δppm 7.42-7.53(m,2H)8.04-8.14(m,2H)；MS m/z 165.2(M+H)⁺；HPLC ＞ 99.5%, RT= 1.96 minute.

By 5- (4- fluorophenyls) -2H- tetrazoliums (6.61g, 40.3mmol), K₂CO₃(6.68g, 48.3mmol) and iodomethane The mixture of (3.02mL, 48.3mmol) in acetonitrile (115mL) is heated to backflow (actual 82 DEG C), heats one hour.Cold But after, mixture is concentrated to dryness, and residue is distributed between water (75mL) and EtOAc (100mL).Layer is separated, Extract organic layer backward using EtOAc (50mL) and utilize water (50mL) and the organic layer of bittern (50mL) washing combination.In nothing Water MgSO₄Upper dry organic layer, filtering and concentration, to produce the colorless oil that 9.5g solidifies in standing.Pass through flash chromatography point Analysis method purifies residue, to produce 2 kinds of major products：Intermediate 35B as the N2 isomers of white solid：5- (4- fluorobenzene Base) -2- methyl -2H- tetrazoliums (5.09g, 70.9% yield)：Do not observed between methyl and aromatic on 4.42ppm To NOE；¹H NMR(400MHz,DMSO-d₆)δppm 4.42(s,3H)7.33-7.45(m,2H)8.03-8.14(m,2H)；MS m/z 179.2(M+H)⁺；HPLC ＞ 99.5%, RT=1.75 minute.

N1 isomers as white solid：5- (4- fluorophenyls) -1- methyl isophthalic acid H- tetrazoliums (1.87g, 26.1% yield)： The NOE observed between methyl on 4.16ppm and two aromatics on 7.89-7.97ppm confirms the structure ；¹H NMR(400MHz,DMSO-d₆)δppm 4.16(s,3H)7.43-7.53(m,2H)7.89-7.97(m,2H)；MS m/z 179.2(M+H)⁺；HPLC ＞ 99.5%, RT=1.29 minute.

Intermediate 35C and D

By intermediate 35B (5- (4- fluorophenyls) -2- methyl -2H- tetrazoliums, 1g, 5.61mmol) sulfuric acid (16.45mL, Solution in 309mmol) is cooled to 0 DEG C, and fuming nitric aicd (0.288mL, 6.17mmol) is then added dropwise.2.5 hours it Afterwards, more fuming nitric aicds (0.065mL, 1.403mmol) are added and allow mixture to be heated to 20 DEG C.After 5 hours, will Mixture pours into 2：In 1 mixture of ice and water (150mL), cause the formation of white suspension.After 30 minutes, filter solid is crossed, Using water washing (4 × 10mL, until the neutral pH of washing), under high vacuum, in 25 DEG C of dryings, until constant weight：As ash 5- (the fluoro- 3- nitrobenzophenones of 4-) -2- methyl -2H- tetrazoliums (1.16g, 93% yield) of white solid：¹H NMR(400MHz, DMSO-d₆) δ ppm 4.47 (s, 3H) 7.81 (dd, J=11.2,8.8Hz, 1H) 8.44 (ddd, J=8.7,4.2,2.3Hz, 1H) 8.68 (dd, J=7.2,2.2Hz, 1H)；MS m/z 224.2(M+H)⁺；HPLC 98.3%, RT=1.72 minute.

Solution of the 2- cyanoacetamides (0.888g, 10.56mmol) in DMF (2.268mL) is added to sodium hydride In suspension of the 60%wt mineral oil (0.443g, 11.08mmol) in DMF (5.67mL), to produce grey suspension. (pay attention to after being cooled to 0 DEG C：Hydrogen escapes), stirred 30 minutes in 0 DEG C of mixture by synthesis.Then, 5- (4- are added Fluoro- 3- nitrobenzophenones) the solution of -2- methyl -2H- tetrazoliums (1.15g, 5.15mmol) in DMF (2.3mL), it is dark purple to produce Color solution.After 3 hours, reactant mixture is slowly poured into mixture of ice and water (33.0mL) and concentration HCl (0.952mL) In.The yellow syrup of synthesis is stirred 30 minutes, crosses filter solid, using water washing (3 × 5mL) and and then utilize hexane (2 × 5mL) wash, 40 DEG C of dryings under high vacuum, until constant weight, to produce 2- cyano group -2- (4- (the 2- first as yellow solid Base -2H- tetrazolium -5- bases) -2- nitrobenzophenones) acetamide (1.41g, 95% yield)：¹H NMR(400MHz,DMSO-d₆)δppm 4.49 (s, 3H) 5.77 (s, 1H) 7.77 (s, 1H) 7.95 (d, J=8.2Hz, 1H) 8.03 (s, 1H) 8.51 (dd, J=8.2, 1.8Hz, 1H) 8.70 (d, J=1.8Hz, 1H)；MS m/z 288.1(M+H)⁺；HPLC 96.4%@220nm, RT=1.31 point Clock.

By ferric chloride hexahydrate (2.82g, 10.44mmol) and zinc (2.276g, 34.8mmol) be added in batches 2- cyano group- 2- (4- (2- methyl -2H- tetrazolium -5- bases) -2- nitrobenzophenones) acetamide (1g, 3.48mmol) is in DMF (4.75mL) and water In the mixture of (4.75mL), to produce yellow suspension, 100 DEG C are heated to, is heated 1.25 hours.Then, will mix Thing is cooled to 20 DEG C, is diluted using MeOH (50.0mL), filters over celite and is concentrated into actual 20mL under reduced pressure (to go Except most MeOH).Then, using water (50mL) and EtOAc (100mL) diluted mixture, consumingly stir and filter.Profit With EtOAc (2 × 50mL) aqueous layer extracteds and utilize saturation NaHCO₃The organic layer that the washing of (50mL) and bittern (30mL) merges. Anhydrous MgSO₄Upper dry organic layer, filtering and concentration, should using the purifying of fast chromatographic method to produce 489mg violet solids Solid, with provide violet solid 2- amino -6- (2- methyl -2H- tetrazolium -5- bases) -1H- indoles -3- formamides (356mg, 39.7% yield)：¹H NMR(400MHz,DMSO-d₆)δppm 4.38(s,3H)6.57(s,2H)7.01(s,2H)7.61-7.69 (m,2H)7.81(s,1H)10.77(s,1H)；MS m/z 258.2(M+H)⁺；HPLC ca.78%, RT=1.34 minutes.

By intermediate 35D (2- amino -6- (2- methyl -2H- tetrazolium -5- bases) -1H- indoles -3- formyls in microwave tube Amine, 0.35g, 1.361mmol), the methoxy in methyl 2- phenylacetates (0.288mL, 2.041mmol) and MeOH (0.467mL) The mixture of base sodium 25%wt. and methanol (3.03mL) is placed in micro-wave oven, and is heated to 140 DEG C, is heated one hour.Cold But to after room temperature, diluted, stirred the mixture for 30 minutes, to allow to crystallize using water (1mL) and AcOH (4mL).Filtering is solid Body, washed using MeOH (5 × 1mL), under high vacuum, in 40 DEG C of dryings, until constant weight, to provide brown solid 2- benzyls Base -7- (2- methyl -2H- tetrazolium -5- bases) -9H- pyrimidos [4,5-b] indoles -4- alcohol (220mg, 45.2% yield).¹H NMR (400MHz,DMSO-d₆) δ ppm 4.03 (s, 2H) 4.43 (s, 3H) 7.24-7.29 (m, 1H) 7.34 (t, J=7.8Hz, 2H) 7.37-7.43 (m, 2H) 7.92 (dd, J=8.0,1.4Hz, 1H) 8.04-8.10 (m, 2H) 12.38 (s, 1H) 12.47 (s, 1H)； MS m/z 358.2(M+H)⁺；HPLC 82.9%, RT=1.89 minute.

In 2-5mL microwave vials, crude product 2- benzyls -7- (2- methyl -2H- tetrazolium -5- bases) -9H- pyrimidos are added [4,5-b] indoles -4- alcohol (0.220g, 0.616mmol) and POCl₃(3.90mL, 41.9mmol), to provide brown suspension. Vial is placed in micro-wave oven, and is heated to 175 DEG C, heats 15 minutes, then allows to cool down.Then, it is reaction is mixed Compound pours into water and ice mixture (80mL), by NaOH 50%wt (11mL) and and then EtOAc (80mL) be slowly added Alkalize to pH8.Filter some solids and separate layer.Using EtOAc (80mL) aqueous layer extracteds and in anhydrous MgSO₄Upper drying has Machine layer, filter and be concentrated to dryness, to provide corresponding chlorinated derivative：Chloro- 7- (the 2- of 2- benzyls -4- as brown solid Methyl -2H- tetrazolium -5- bases) -9H- pyrimidos [4,5-b] indoles (189mg, 82% yield).¹H NMR(400MHz,DMSO-d₆) δ ppm 4.31 (s, 2H) 4.46 (s, 3H) 7.20-7.26 (m, 1H) 7.28-7.39 (m, 4H) 8.09 (dd, J=8.2,1.2Hz, 1H) 8.21-8.25 (m, 1H) 8.39 (d, J=8.2Hz, 1H) 12.93 (s, 1H)；MS m/z 376.2(M+H)⁺；HPLC 95.6%, RT=2.30 minute.

In micro-wave oven, at 140 DEG C, by the chloro- 7- of the 2- benzyls -4- (2- methyl -2H- tetrazoliums -5- of foregoing description preparation Base) -9H- pyrimidos [4,5-b] indoles (0.050mg, 0.133mmol) and Et₃N (0.037mL, 0.266mmol) and 3- (piperazines Pyridine -1- bases) mixture of the propane -1- amine (0.033mL, 0.200mmol) in MeOH (0.6mL) heat 25 minutes.Cooling down After the evaporation of solvent, residue (MeOH- water (0.5%TFA) 20% to 100%MeOH) is purified by RP-HPLC to provide 55mg embodiment 35：2- benzyls -7- (2- methyl -2H- tetrazolium -5- bases)-N- (3- (piperidin-1-yl) propyl group) -9H- pyrimidos [4,5-b] indoles -4- amine 2,2,2- triflutates：¹H NMR(400MHz,DMSO-d₆)δppm 1.27-1.41(m,1H) 1.53-1.72 (m, 3H) 1.79 (d, J=13.69Hz, 2H) 1.98-2.09 (m, 2H) 2.75-2.87 (m, 2H) 3.09 (dt, J= 10.27,5.23Hz, 2H) 3.39 (d, J=11.35Hz, 2H) 3.69 (q, J=5.87Hz, 2H) 4.09 (s, 2H) 4.44 (s, 3H) (7.18-7.24 m, 1H) 7.31 (t, J=7.63Hz, 2H) 7.35-7.42 (m, 2H) 7.51 (br.s., 1H) 7.93 (dd, J= 8.22,1.17Hz, 1H) 8.11 (d, J=1.17Hz, 1H) 8.43 (d, J=8.22Hz, 1H) 9.04 (br.s., 1H) 12.15 (s, 1H)；HPLC 99%at 254nm, Rt 2.063 minutes；HRMS m/z 482.2817(M+H)+.

Embodiment 36 (the Jia oxadiazoles from cyanide)

Hydroxylamine hydrochloride (32.7mg, 0.471mmol) is added to (2- benzyls -4- (3- (piperidin-1-yl) third of embodiment 37 Base) amino) -9H- pyrimidos [4,5-b] nitrile of indoles -7,50mg, 0.118mmol) in solution in EtOH (1.5mL), afterwards DIPEA (84 μ L, 0.483mmol) is added, to produce light yellow suspension.It is stirred at room temperature 2.5 days and stirs 6 at 75 DEG C After hour, evaporation solvent and addition water (3mL) and after 30min stir, collect solid, using water (3 × 1mL) washing with Under high vacuum, in 35 DEG C of drying solid materials, until constant weight, (Z) -2- benzyls-N ' to produce as Tan solid - Hydroxyl 4- (3- (piperidin-1-yl) propyl group) amino)-9H- pyrimidos [4, the 5-b] carboximidamide of indoles-7-HCl (53mg, 0.107mmol, 91% yield)；¹H NMR(400MHz,DMSO-d₆)δppm 1.58-1.85(m,6H)1.98-2.10(m,2H) 2.69-2.90(m,2H)2.94-3.15(m,2H)3.34-3.46(m,2H)3.59-3.74(m,2H)4.05(s,2H)5.84 (br.s., 2H) 7.15-7.24 (m, 1H) 7.29 (t, J=7.4Hz, 3H) 7.37 (d, J=7.4Hz, 2H) 7.55 (dd, J= 8.2,1.2Hz, 1H) 7.72 (d, J=1.2Hz, 1H) 8.25 (d, J=8.6Hz, 1H) 9.47 (d, J=7.4Hz, 1H) 9.56 (s, 1H)11.88(s,1H)；HRMS m/z 458.2662(M+H)⁺.HPLC 95.4%@220nm and 97.4%@254nm, RT= 1.40 minute.

Acetic anhydride (0.917mL, 9.72mmol) is added to (Z) -2- benzyls-N '-hydroxyl 4- (3- (piperidin-1-yl) Propyl group) amino) -9H- pyrimidos [4,5-b] carboximidamide of indoles -7, in HCl (0.040g, 0.081mmol), to produce Yellow-brown suspension, and 140 DEG C are heated the mixture to by microwave, heat 30 minutes.Then, evaporation solvent, and pass through Fast chromatographic method purifies residue, the 1- (2- benzyl -7- (5- as Tan solid to produce 36mg (85% yield) Methyl isophthalic acid, 2,4- di azoly -3- bases) -4- ((3- (piperidin-1-yl) propyl group) amino) -9H- pyrimidos [4,5-b] indoles -9- Base) ethyl ketone, is dissolved in methanol (2.3mL), and utilize DBU (0.021mL, 0.138mmol) processing immediately.By the yellow of synthesis Solution is heated to flowing back, and heats 30 minutes, is subsequently cooled to 0 DEG C, while stir 1 hour.Filter solid is crossed, and utilizes cold MeOH (2 × 0.5mL) is washed, under high vacuum, 40 DEG C of dryings, until constant weight, to provide the embodiment 36 as Tan solid： 2- benzyls -7- (5- methyl isophthalic acids, 2,4- di azoly -3- bases)-N- (3- (piperidin-1-yl) propyl group) amino) -9H- pyrimidos [4,5- B] indoles -4- amine (20mg, 51.3% yield)：¹H NMR(400MHz,DMSO-d₆) δ ppm 1.38 (m, J=4.7Hz, 2H) 1.50 (five one group, J=5.5Hz, 4H) 1.80 (five one group, J=6.9Hz, 2H) 2.18-2.45 (m, 6H) 2.67 (s, 3H) 3.57-3.70 (m, 2H) 4.04 (s, 2H) 7.15-7.22 (m, 1H) 7.27 (m, J=7.6,7.6Hz, 2H) 7.34 (t, J= 5.9Hz, 1H) 7.36-7.40 (m, 2H) 7.83 (dd, J=8.2,1.4Hz, 1H) 8.01 (d, J=1.4Hz, 1H) 8.39 (d, J= 8.2Hz,1H)12.02(br.s.,1H)；HRMS m/z 482.2663(M+H)⁺.HPLC 99.3%, RT=1.79 minutes.

Embodiment 37

In microwave tube, by 2- amino -6- cyanogen -1H- indoles -3- formamides (0.172g, 0.859mmol), methyl 2- second Sodium methoxide 30%wt (0.403mL, 2.148mmol) in acid phenenyl ester (0.303mL, 2.148mmol) and MeOH is in methanol Orange mixture in (2.82mL) heats 45 minutes at 140 DEG C.Then, add 2- phenylacetates (0.151mL, The new load of sodium methoxide 30%wt (0.201mL, 1.074mmol) 1.074mmol) and in MeOH and by vial It is placed in microwave, and is heated 45 minutes at 140 DEG C again.Then, after cooling to room-temperature, AcOH is added (0.197mL, 3.44mmol) and stirred 1 hour in 20 DEG C of slurries by synthesis.Filter solid is crossed, is washed using MeOH (3 × 1mL) And under a high vacuum, dried at 20 DEG C, until constant weight, to produce the intermediate 37B as Tan solid：2- benzyls -4- Hydroxyl -9H- pyrimidos [4,5-b] indoles -7- nitriles (182mg, 70.5% yield)：¹H NMR(400MHz,DMSO-d₆)δppm 4.03 (s, 2H) 7.22-7.29 (m, 1H) 7.30-7.36 (m, 2H) 7.36-7.43 (m, 2H) 7.58 (dd, J=8.2,1.4Hz, 1H) 7.82-7.87 (m, 1H) 8.05 (d, J=8.2Hz, 1H) 12.59 (br.s., 2H)；MS m/z 301.2(M+H)⁺；HPLC The@254nm of 94.2%@220nm and 91.3%；RT=1.90 minutes.

By 2- benzyls -4- hydroxyl -9H- pyrimidos [4,5-b] indoles -7- nitriles (intermediate 37B, 0.180g, 0.599mmol) 95 DEG C are heated to the red mixture of phosphorous oxychloride (3.63mL, 39.0mmol), and is stirred 16 hours.In Rotary Evaporators On be concentrated to dryness after, by the peony foam suspension of synthesis in saturation NaHCO₃In (10mL), and stir 30 minutes.Collect Solid and utilization water (3 × 1mL) washing, under a high vacuum, are dried at 40 DEG C, until constant weight, are consolidated using producing as yellowish-brown The intermediate 37C of body：Chloro- 9H- pyrimidos [4, the 5-b] indoles -7- nitriles of 2- benzyls -4- (190mg, 99% yield), immediately by it For next step：MS m/z 319.2(M+H)⁺；95.0%@220nm and of HPLC 92.3%@254nm, RT=2.28 Minute.

By microwave, by chloro- 9H- pyrimidos [4, the 5-b] indoles -7- nitriles of 2- benzyls -4- (intermediate 37C, 0.190g, 0.596mmol), 3- (piperidin-1-yl) propane -1- amine (0.142mL, 0.894mmol) and triethylamine (0.208mL, 1.490mmol) mixture in MeOH (4.50mL) is heated to 140 DEG C, heats 30 minutes.Then, it is concentrated to dryness, To provide 346mg orange solids, the solid is purified by fast chromatographic method, to provide 176mg yellow solid, by it It is suspended in ether (7mL), and is stirred 1 hour at 20 DEG C.Filter solid is crossed, is washed using ether (3 × 1mL), and in high vacuum Under, dried at 30 DEG C, until constant weight, using provide embodiment 37 as faint yellow solid 2- benzyls -4- ((3- (piperidines - 1- yls) propyl group) amino) -9H- pyrimidos [4,5-b] indoles -7- nitriles (172mg, 68.0% yield)：¹H NMR(400MHz, DMSO-d₆) δ ppm 1.30-1.43 (m, 2H) 1.43-1.57 (m, 4H) 1.80 (m, J=5.5Hz, 2H) 2.18-2.47 (m, 6H) 3.62 (q, J=6.4Hz, 2H) 4.04 (s, 2H) 7.15-7.22 (m, 1H) 7.27 (m, J=7.4,7.4Hz, 2H) 7.33-7.39 (m, 2H) 7.47 (t, J=5.7Hz, 1H) 7.62 (dd, J=8.2,1.2Hz, 1H) 7.79 (d, J=1.2Hz, 1H) 8.43 (d, J =8.2Hz, 1H) 12.19 (s, 1H)；HRMS m/z 425.2448(M+H)⁺；HPLC ＞ 99%, RT=1.68 minute.

Embodiment 43

In 2-5mL microwave vials, chloro- 9H- pyrimidos [4,5-b] the indole -7-carboxylic acid's methyl esters of 2- benzyls -4- is added (0.100g, 0.284mmol), (E) -1- (4- (4,4,5,5- tetramethyls -1,3,2-dioxaborolan-2- yls) 3- butane -1- Base) piperidines (0.113g, 0.426mmol), potassium carbonate (0.106g, 0.768mmol) and Pd (Ph3P) 4 (0.05g, 0.044mmol).With N₂(3 vacuum+backfill circulation) purification vial.DME (2.84mL) and water (0.398mL) are added, and With N₂(with vacuum+backfill) rinses vial, then, is heated to 110 DEG C, while stir 24 hours.After cooling, Under reduced pressure, mixture is concentrated to dryness, and residue is purified by fast chromatographic method, pale yellow colored solid is used as to produce (E)-methyl 2- benzyls -4 (4- (piperidin-1-yl)) 1- butane -1- base -9H- pyrimidos [4,5-b] indole -7-carboxylic acid's ester of body (55mg, 0.121mmol, 42.6% yield)：¹H NMR(400MHz,DMSO-d₆)δppm 1.41(s,2H)1.50-1.61(m, 4H)2.30-2.47(m,4H)2.53-2.59(m,2H)2.59-2.70(m,2H)3.91(s,3H)4.27(s,2H)7.16-7.24 (m, 1H) 7.29 (t, J=7.6Hz, 2H) 7.34-7.43 (m, 4H) 7.88 (dd, J=8.2,1.4Hz, 1H) 8.07 (d, J= 1.4Hz, 1H) 8.41 (d, J=8.2Hz, 1H) 12.48 (s, 1H)；HRMS m/z 455.2442(M+H)⁺；The@of HPLC 100% @254nm, the RT=1.84 minutes of 220nm and 99.4%.

Using hydrogen by (4- (the piperidin-1-yl)) 1- butane -1- of (E)-methyl 2- benzyls -4 bases) -9H- pyrimidos [4,5-b] Indole -7-carboxylic acid's ester (20mg, 0.044mmol) and Pd-C 10%wt. (50%wet) (23.41mg) are in MeOH (2mL) and THF Mixture in (2mL) is handled 17 hours.Using DCM (3mL) diluted reaction mixture, filtering, using MeOH (2 × 2mL) and Then rinsed, and be concentrated to dryness using DCM (2 × 2mL), to provide 19mg faint yellow solid, pass through flash chromatography point Analysis method purifies the solid-state, to produce white solid (14mg), utilizes CH₃CN (2mL) handles the white solid.At 20 DEG C by white After suspension stirs 1 hour, filter solid is crossed, utilizes CH₃CN (1 × 1mL) is washed and dried under a high vacuum at 40 DEG C, Until constant weight, to provide the compound as the embodiment 43 of white solid, 2- benzyls -4 (4- (piperidin-1-yl)) butyl) - 9H- pyrimidos [4,5-b] indole -7-carboxylic acid's methyl esters (14.4mg, 71.7% yield)：¹H NMR(400MHz,DMSO-d₆)δppm 1.36 (m, J=5.5Hz, 2H) 1.45 (five one group, J=5.5Hz, 4H) 1.57 (five one group, J=7.3Hz, 2H) 1.83 (dt, J=14.9,7.4Hz, 2H) 2.18-2.31 (m, 6H) 3.20-3.28 (m, 2H) 3.91 (s, 3H) 4.26 (s, 2H) 7.16- 7.22 (m, 1H) 7.28 (t, J=7.4Hz, 2H) 7.32-7.38 (m, 2H) 7.90 (dd, J=8.2,1.2Hz, 1H) 8.09 (d, J =1.2Hz, 1H) 8.25 (d, J=8.2Hz, 1H) 12.48 (br.s., 1H)；HRMS m/z 457.2598(M+H)⁺；HPLC ＞ 99.5%, RT=1.75 minute.

Embodiment 44

At 140 DEG C, in micro-wave oven, by chloro- 9H- pyrimidos [4,5-b] the indole -7-carboxylic acid's salt of 2- benzyls -4 (0.100g, 0.284mmol)、Et₃N (0.079mL, 0.569mmol) and the tert-butyl group (3- aminopropyls) (methyl) methyl carbamate The mixture of (0.080g, 0.426mmol) in MeOH (1mL) heats 40 minutes.Solvent is removed under reduced pressure, and by fast Fast red, orange, green, blue, yellow (ROGBY) purifies residue, and to provide 0.092mg thick Boc derivatives, it is directly used next step.By TFA (1.0ml, 12.98mmol) is added dropwise to 2- benzyls -4 ((3- tertbutyloxycarbonyls) (methyl) amino) propyl group) amino) -9H- is phonetic Pyridine is simultaneously in the cold suspension of [4,5-b] indole -7-carboxylic acid's methyl esters (0.092g, 0.183mmol), and allow mixture at 30 points Warmed to room temperature on clock.After using dilution with toluene, solvent is removed under reduced pressure, then, dilutes residue using EtOAc, with Produce the solid that 85mg is directly used in next step：HRMS m/z 404.2091(M+H)+.

In acetone (0.2mL), ((3- methylaminos) propyl group) amino of 2- benzyls -4 is heated at 70 DEG C) -9H- pyrimidos [4, 5-b] 2,2,2- trifluoroacetic acid methyl ester (0.020g, 0.039mmol) of indole -7-carboxylic acid's salt, sodium carbonate (8.81mg, 0.083mmol), sodium iodide (1.448mg, 9.66 μm of ol) and 2- (2- (2- Chloroethoxies) ethyoxyl) ethanol (6.46 μ l, Mixture 0.044mmol).After 15 hours, addition 2- (2- (2- Chloroethoxies) ethyoxyl) ethanol (6.46 μ l, Part II 0.044mmol), and again heat mixture 15 hours at 70 DEG C.After cooling to room-temperature, utilize EtOAc diluted mixtures, using water washing, in anhydrous MgSO₄Upper drying, filtering, and evaporation solvent, to provide residue, pass through Fast chromatographic method purifies, to provide 9mg embodiment 44：¹H NMR(400MHz,DMSO-d₆)δppm 1.74-1.85(m, 2H) 2.25 (br.s., 3H) 2.55 (br.s., 2H) 3.32-3.36 (m, 4H) 3.39-3.46 (m, 6H) 3.51 (t, J= 5.87Hz, 2H) 3.64 (q, J=6.52Hz, 2H) 3.88 (s, 3H) 4.04 (s, 2H) 4.53 (br.s., 1H) 7.18 (t, J= 7.40Hz, 1H) 7.28 (t, J=7.63Hz, 2H) 7.37 (d, J=7.04Hz, 2H), 7.55 (t, J=5.28Hz, 1H) 7.82 (dd, J=8.22,1.17Hz, 1H) 7.99 (s, 1H) 8.27 (d, J=8.22Hz, 1H) 12.05 (s, 1H)；HRMS m/z 536.2855(M+H)⁺；HPLC RT 2.035 minutes.

Embodiment 45 and 51

In 2-5mL microwave vials, the chloro- 9H- of 2- benzyls -4- of (2.000mL, 49.4mmol) are added in MeOH Pyrimido [4,5-b] indole -7-carboxylic acid's methyl esters (0.050g, 0.142mmol) and N1- (3- aminopropyls)-N1- methylpropane -1, 3- diamines (0.11 5mL, 0.711mmol), to provide yellow-brown suspension.Vial is placed in microwave, and heated To 140 DEG C, 30min is heated.After 30 minutes, mixture is concentrated to dryness on a rotary evaporator, and passes through quick color Analytic approach purifying residue is composed, and from CH₃CN is freezed, to provide two kinds of different products：Reality as the product of list-N- alkylations Apply example 45：4- ((3- ((3- aminopropyls) (methyl) amino) propyl group) amino) 2- benzyl -9H- pyrimidos as white solid [4,5-b] indole -7-carboxylic acid's methyl esters (44mg, 67.2% yield)；¹H NMR(400MHz,DMSO-d₆) δ ppm 1.44 (dt, J= 13.8,6.6Hz, 2H) 1.72 (dt, J=13.7,6.8Hz, 2H) 2.11 (s, 3H) 2.24-2.30 (m, 2H) 2.33 (t, J= 6.7Hz,2H)2.47(br.s.,2H)3.51-3.61(m,2H)3.76-3.85(m,3H)3.97(s,2H)7.08-7.15(m, 1H) 7.17-7.24 (m, 2H) 7.27-7.34 (m, 2H) 7.50 (t, J=5.3Hz, 1H) 7.76 (dd, J=8.2,1.6Hz, 1H) 7.92 (d, J=1.6Hz, 1H) 8.20 (d, J=8.2Hz, 1H)；MS m/z 461.2(M+H)⁺；HPLC ＞ 99%, RT=1.63 Minute.

Embodiment 51 as the product of di-alkyl：4,4 '-(((the MU diyl) two as faint yellow solid (propane -3,1- diyls)) two (urea diyls)) two (2- benzyl -9H- pyrimido [4,5-b] indole -7-carboxylic acids dimethyl esters) (3.7mg, 6.71% yield)：¹H NMR(400MHz,DMSO-d₆)δppm 1.18-1.29(m,4H)1.79-1.91(m,4H) 2.25 (s, 3H) 3.58-3.71 (m, 4H) 3.84 (s, 6H) 3.97 (s, 4H) 7.07-7.16 (m, 2H) 7.22 (t, J=7.4Hz, 4H) 7.29-7.34 (m, 4H) 7.53 (t, J=5.3Hz, 2H) 7.77 (dd, J=8.2,1.4Hz, 2H) 7.96 (d, J=1.4Hz, 2H) 8.22 (d, J=8.2Hz, 2H) 12.01 (s, 2H)；MS m/z 776.3(M+H)⁺；The@220nm and of HPLC 94.6% 93.8%@254nm, RT=2.01 minutes.

Embodiment 52

By 2,5- dioxo pyrrolidin -1- bases -5- ((3aS, 4S, 6aR) -2- oxos hexahydro -1H- thiophene [3,4-d] miaows Azoles -4- bases) valerate (12.01mg, 0.035mmol) is added to 4- ((3- ((3- aminopropyls) (methyl) amino) propyl group) amino) 2- benzyl -9H- pyrimidos [4,5-b] indole -7-carboxylic acid's methyl esters (embodiment 45,15mg, 0.033mmol) and triethylamine (6.81 μ L, 0.049mmol) in solution in DMF (750 μ L, 9.69mmol), to produce yellow solution.Stirred 1 hour at 20 DEG C Afterwards, mixture is concentrated to dryness, and residue is purified by fast chromatographic method, to provide weak yellow foam.Will bubble Foam is suspended in Et₂In O (1mL), and stir 30 minutes, and collect solid, utilize Et₂O (2 × 0.5mL) is washed and in high vacuum Under, in 20 DEG C of dryings, until constant weight, to produce the compound as the embodiment 52 of yellow solid：2- benzyls -4- ((3- (first Base) (3- (5- ((3aS, 4S, 6aR) -2- oxos hexahydro -1H- thiophene [3,4-d] imidazol-4 yl) pentane amino) propyl group) amino) Propyl group) amino) -9H- pyrimidos [4,5-b] indole -7-carboxylic acid's methyl esters (17mg, 76% yield)：¹H NMR(400MHz,DMSO- d₆) δ ppm 1.17-1.34 (m, 3H) 1.36-1.51 (m, 2H) 1.51-1.64 (m, 3H) 1.78 (dt, J=13.5,6.6Hz, 2H) 2.02 (t, J=7.4Hz, 2H) 2.17 (s, 3H) 2.32 (t, J=7.0Hz, 2H) 2.40 (t, J=6.7Hz, 2H) 2.55 (d, J=12.3Hz, 1H) 2.77 (dd, J=12.3,5.1Hz, 1H) 2.99-3.11 (m, 3H) 3.58-3.68 (m, 2H) 3.88 (s, 3H) 4.04 (s, 2H) 4.08 (m, J=4.9,4.9,2.3Hz, 1H) 4.26 (dd, J=7.6,5.3Hz, 1H) 6.34 (s, 1H) 6.40 (s, 1H) 7.14-7.22 (m, 1H) 7.27 (t, J=7.4Hz, 2H) 7.37 (d, J=7.0Hz, 2H) 7.54 (t, J= 5.5Hz, 1H) 7.74 (t, J=5.5Hz, 1H) 7.82 (dd, J=8.2,1.6Hz, 1H), 7.99 (d, J=1.6Hz, 1H) 8.27 (d, J=8.2Hz, 1H) 12.05 (s, 1H)；MS m/z 687.3(M+H)⁺；HPLC ＞ 99.5%, RT=1.70 minute.

Embodiment 53

Intermediate 53A is prepared from 2 (3- tolyls) ethyl acetates of business.Then, as retouched for embodiment 15,45 and 47 State, be converted into intermediate 53B.Then, azirine part is developed according to the description provided for embodiment 25.Finally The step of be based on embodiment 52.

Make 2- (tolyl) ethyl acetate (4.8g, 26.9mmol), NBS (5.27g, 29.6mmol) and benzoyl peroxide first The mixture of acyl (0.110g, 0.454mmol) is in CCl₄Flowed back in (28mL).After 5 hours, reaction is cooled to 5 DEG C, mistake Filter and removal solvent.Using ethylacetate-hexane, the corresponding benzyl bromides of 3.8g are produced by the purifying of fast chromatographic method Derivative：¹H NMR (400MHz, DMSO-d6) δ ppm 1.19 (t, J=6.70Hz, 3H) 3.67 (s, 2H) 4.09 (q, J= 6.70Hz,2H)4.69(s,2H)7.15-7.25(m,1H)7.27-7.42(m,3H)；The material is directly used in next step Suddenly.

By 2- (3- (bromomethyl) phenyl) ethyl acetates (8.11g, 31.5mmol) and 4- methylmorpholine 4- oxide water Mixture of the compound (5.54g, 47.3mmol) in the dioxane of Isosorbide-5-Nitrae-(110mL) is heated to 100 DEG C, heats 1.5 hours.Cold But to after room temperature, the volume of solvent is reduced to half, and then utilize Et₂O: EtOAc (1: 1,120mL) dilutes, and utilizes Water (50mL) washs and utilized EtOAc (60mL) aqueous layer extracted.Using water (2 × 50mL), then, washed using bittern (50mL) The organic layer of merging.In anhydrous MgSO₄Upper dry organic layer, filtering and concentration, to produce the faint yellow oil of 4.72g, by quick Red, orange, green, blue, yellow (ROGBY) is purified, and faint yellow oily 2- (3- formylphenyls) acetate (2.91g, 48% yield) is used as to provide：¹H NMR (400MHz, DMSO-d6) δ ppm 1.19 (t, J=7.0Hz, 3H) 3.81 (s, 2H) 4.09 (q, J=7.0Hz, 2H) 7.52-7.65(m,2H)7.79-7.85(m,2H)10.00(s,1H)；MS m/z 207.2(M+H)⁺；HPLC 99%, RT= 1.67 minute.

Trimethyl (trifluoromethyl) silane (3.13mL, 21.20mmol) is added to and is cooled to 0-5 DEG C of ethyl 2- (3- Formylphenyl) acetate (2.91g, 15.14mmol) and cesium fluoride (0.161g, 1.060mmol) be in DMF (20.19mL) In mixture.After stirring 1.5 hours, solution of the addition TBAF 1M in THF (15.14mL).Produced in 0-5 DEG C of stirring Yellow solution, and after 30 minutes, pour the mixture into water (150mL) and using MTBE (1 × 150mL, then 2 × 100mL) extract.Using water (1 × 150mL, then 1 × 100mL) and the organic layer of bittern (100mL) washing combination, and then, In anhydrous MgSO₄Upper dry organic layer, filtering and concentration, it is pure by fast chromatographic method to produce 3.84g light orange oil Change, colourless oily 2- (3- (2,2,2- tri- fluoro- 1- ethoxys) phenyl) ethyl acetate (663mg, 16% production is used as to produce Amount)：¹H NMR (400MHz, DMSO-d6) δ ppm 1.17 (t, J=7.0Hz, 3H) 3.68 (s, 2H) 4.08 (q, J=7.0Hz, 2H) 5.13 (q, J=7.4Hz, 1H) 6.82 (s, 1H) 7.24-7.31 (m, 1H) 7.37 (t, J=7.2Hz, 3H)；MS m/z 263.1(M+H)⁺；@220nm, the RT=1.82 minutes of HPLC 93.7%.

Benzyl bromide a-bromotoluene (0.330mL, 2.78mmol) is added to 2- (3- (2,2,2- tri- fluoro- 1- ethoxys) phenyl) acetic acid second Ester (0.648g, 2.471mmol) and K₂CO₃In the mixture of (1.059g, 7.66mmol) in acetonitrile (17.00mL), and stir Mixture is mixed, while is heated to flowing back (75-80 DEG C), 24 hours.After cooling down and being concentrated to dryness, pass through flash chromatography point Analysis method purifies residue, to produce intermediate 53A：2- (3- (1- (benzyloxy) -2,2,2- trifluoroethyls) benzene as colorless oil Base) ethyl acetate (686mg, 79% yield)：MS m/z 353.2(M+H)⁺；HPLC 99.7%, RT=2.19 minute.

Add intermediate 15B (0.310g, 1.329mmol), 2- (3- (1- (benzyloxy) -2,2,2- trifluoroethyls) phenyl) Sodium methoxide 30%wt. in ethyl acetate (0.679g, 1.927mmol) and intermediate 53A and MeOH (0.524mL) is in methanol The mixture of (3.23mL), to produce thin brown suspension, 140 DEG C are heated in microwave device, heating 1 is small When.After cooling down and being diluted using MeOH (0.75mL) and AcOH (0.167mL, 2.92mmol), at 20 DEG C, hanged by caused by Supernatant liquid stirs 2 hours.Then, solid is collected, and using MeOH (4 × 0.5mL) washings, then under high vacuum, 40 DEG C drying, until constant weight, 2- (3- (1- (benzyloxy) -2,2,2- trifluoroethyls) benzyl) to provide as Tan solid - 4- hydroxyl -9H- pyrimidos [4,5-b] indole -7-carboxylic acid's methyl esters (399mg, 57.6% yield)：¹H NMR(400MHz,DMSO- D6) δ ppm 3.87 (s, 3H) 4.09 (s, 2H) 4.45-4.57 (m, 2H) 5.23 (q, J=7.2Hz, 1H) 7.20-7.29 (m, 5H) 7.36-7.51 (m, 3H) 7.52 (s, 1H) 7.84 (dd, J=8.2,1.4Hz, 1H) 8.01 (d, J=1.4Hz, 1H) 8.03 (d, J =8.2Hz, 1H) 12.46 (br.s., 1H) 12.56 (br.s., 1H)；MS m/z 522.2(M+H)⁺；The@of HPLC 92.7% @254nm, the RT=2.20 minutes of 220nm and 91.7%.

By 2- (3- (1- (benzyloxy) -2,2,2- trifluoroethyls) benzyl) -4- hydroxyl -9H- pyrimidos [4,5-b] indoles - Mixture of the 7- carboxylate methyl esters (0.395g, 0.757mmol) in phosphorous oxychloride (6mL, 64.4mmol) is heated to 90 DEG C, adds Heat 2 hours, then after cooling, is concentrated to dryness, to produce 650mg brown foam, is suspended in saturation NaHCO₃In (15mL), and stir 1 hour.Filter solid is crossed, is washed using water (3 × 2mL) and under a high vacuum, is done at 40 DEG C It is dry, until constant weight, 2- (3- (1- (benzyloxy) -2,2,2- trifluoroethyls) benzyl) -4- to provide as Tan solid are chloro- 9H- pyrimidos [4,5-b] indole -7-carboxylic acid's methyl esters (375mg, 92% yield) and equally it is used for next step：MS m/z 540.2(M+H)⁺；HPLC 92%, RT 2.51 minutes.

In micro-wave oven, by 2- (3- (1- (benzyloxy) -2,2,2- trifluoroethyls) benzyl) chloro- 9H- pyrimidos of -4- [4, 5-b] indole -7-carboxylic acid's methyl esters (0.375g, 0.695mmol) and N1- (3- aminopropyls)-N1- methylpropane -1,3- diamines The mixture of (0.784mL, 4.86mmol) in MeOH (9.83mL, 243mmol) is heated to 140 DEG C, heats 30 minutes.So Afterwards, after being concentrated to dryness under reduced pressure, by brown oil caused by the purifying of fast chromatographic method, steeped using providing as yellow 4- ((3- ((3- aminopropyls) (methyl) amino) propyl group) amino) -2- (3- (1- (benzyloxy) -2,2,2- trifluoroethyls) benzyls of foam Base) -9H- pyrimidos [4,5-b] indole -7-carboxylic acid's methyl esters：¹H NMR (400MHz, DMSO-d6) δ ppm 1.48 (dt, J= 14.0,6.9Hz, 2H) 1.75 (five one group, J=6.7Hz, 2H) 2.14 (s, 3H) 2.24-2.42 (m, 4H) 2.52-2.60 (m, 2H) 3.61 (q, J=6.3Hz, 2H) 3.88 (s, 3H) 4.10 (s, 2H) 4.49 (s, 2H) 5.17 (q, J=7.0Hz, 1H) 7.18- (7.30 m, 5H) 7.31-7.36 (m, 1H) 7.39 (t, J=7.6Hz, 1H) 7.44-7.53 (m, 2H) 7.58 (t, J=5.3Hz, 1H) 7.84 (dd, J=8.2,1.4Hz, 1H) 8.00 (d, J=1.4Hz, 1H) 8.28 (d, J=8.2Hz, 1H)；MS m/z 649.3(M+H)⁺；@254nm, the RT=1.96 minutes of 97.6%@220nm and of HPLC 95.5%.

Solution of the di-t-butyl-sodium acid carbonate (0.124mL, 0.533mmol) in DCM (1mL) is slowly added into 4- ((3- ((3- aminopropyls) (methyl) amino) propyl group) amino) -2- (3- (1- (benzyloxy) -2,2,2- trifluoroethyls) benzyl) - 9H- pyrimidos [4,5-b] indole -7-carboxylic acid's methyl esters (0.288g, 0.444mmol) and triethylamine (0.074mL, 0.533mmol) In mixture in DCM (3mL) and MeOH (2mL), to produce yellow solution.After 20 DEG C are stirred 45 minutes, by solution It is concentrated to dryness, to produce 373mg yellow colored foams, the foam is purified by fast chromatographic method, is steeped using providing as yellow Intermediate 53B, 2- (3- (1- (benzyloxy) -2,2,2- trifluoroethyls) benzyl) -4- ((3- ((3- ((tertbutyloxycarbonyl) ammonia of foam Base) propyl group) (methyl) amino) propyl group) amino) -9H- pyrimidos [4,5-b] indole -7-carboxylic acid's methyl esters (306mg, 92% production Amount)：¹H NMR (400MHz, DMSO-d6) δ ppm 1.33 (s, 9H) 1.52 (dt, J=14.1,7.0Hz, 2H) 1.67-1.81 (m, 2H) 2.12 (s, 3H) 2.28 (t, J=7.2Hz, 2H) 2.34 (t, J=6.8Hz, 2H) 2.92 (q, J=6.7Hz, 2H) 3.54-3.67 (m, 2H) 3.88 (s, 3H) 4.10 (s, 2H) 4.49 (s, 2H) 5.17 (q, J=6.8Hz, 1H) 6.76 (t, J= 5.5Hz, 1H) 7.16-7.30 (m, 5H) 7.31-7.36 (m, 1H) 7.39 (t, J=7.6Hz, 1H) 7.43-7.51 (m, 2H) 7.53 (t, J=5.3Hz, 1H) 7.83 (dd, J=8.4,1.4Hz, 1H) 8.00 (d, J=1.4Hz, 1H) 8.27 (d, J=8.2Hz, 1H) 12.06(s,1H)；MS m/z 749.3(M+H)⁺；HPLC 97.7%, RT=2.06 minute.

At 20 DEG C, using hydrogen by 2- (3- (1- (benzyloxy) -2,2,2- trifluoroethyls) benzyl) -4- ((3- ((3- ((tertiary fourths Oxygen carbonyl) amino) propyl group) (methyl) amino) propyl group) amino) -9H- pyrimidos [4,5-b] indole -7-carboxylic acid's methyl esters (0.306g, 0.409mmol) and Pd-C 10%wt (50%wet) (0.304g, 0.143mmol) are in MeOH (9.92mL) Mixture is handled 22 hours.Then, reactant mixture is filtered over celite, using MeOH (2 × 10mL) and utilizes DCM: MeOH (1: 1,2x 10mL) rinses block, is then concentrated to dryness on a rotary evaporator, to produce 226mg oil, by fast Fast red, orange, green, blue, yellow (ROGBY) purifying, to provide the 4- ((3- ((3- ((tertbutyloxycarbonyl) amino) propyl group) (methyl) as white foam Amino) propyl group) amino) -2- (the fluoro- 1- ethoxys of 3-2,2,2- tri-) benzyl) -9H- pyrimidos [4,5-b] indole -7-carboxylic acid's first Ester (202mg, 75% yield)：¹H NMR(400MHz,DMSO-d6)δppm 1.33(s,9H)1.48-1.62(m,2H)1.71- 1.85 (m, 2H) 2.16 (s, 3H) 2.25-2.35 (m, 2H) 2.39 (t, J=6.8Hz, 2H) 2.86-3.00 (m, 2H) 3.56- 3.70 (m, 2H) 3.88 (s, 3H) 4.06 (s, 2H) 5.01-5.14 (m, 1H) 6.77 (m, J=6.3Hz, 2H) 7.30 (d, J= 4.7Hz, 2H) 7.36-7.42 (m, 1H) 7.49 (s, 1H) 7.51-7.57 (m, 1H) 7.82 (dd, J=8.2,1.2Hz, 1H) 7.99 (d, J=1.2Hz, 1H) 8.26 (d, J=8.2Hz, 1H) 12.06 (br.s., 1H)；MS m/z 659.2(M+H)⁺；HPLC ＞ 97%, RT=1.90 minute.

At 20 DEG C, by 4- ((3- ((3- ((tertbutyloxycarbonyl) amino) propyl group) (methyl) amino) propyl group) amino) -2- (3- 2,2,2- tri- fluoro- 1- ethoxys) benzyl) -9H- pyrimidos [4,5-b] indole -7-carboxylic acid's methyl esters (0.200g, 0.304mmol) and Mixture of the high iodine alkane reagent (0.567g, 1.336mmol) of Dai Si-Martin in DCM (7.50mL) stirs one hour.Evaporating To drying, residue is purified by fast chromatographic method, to provide 4- ((3- ((3- ((the tertiary fourth oxygen as yellow colored foam Carbonyl) amino) propyl group) (methyl) amino) propyl group) amino) -2- (3- (2,2,2- trifluoroacetyl groups) benzyl) -9H- pyrimidos [4,5-b] indole -7-carboxylic acid's methyl esters (181mg, 91% yield)：In DMSO-d6¹H NMR are consistent with desired product, still Because the presence of hydrate forms is more complicated：MS m/z 657.3(M+H)⁺；The@220nm of HPLC 96.0% and 95.3%@ (ketone+hydrate) minute of 254nm, RT 1.87 and 1.96.

In micro-wave oven, by 4- ((3- ((3- ((tertbutyloxycarbonyl) amino) propyl group) (methyl) amino) propyl group) amino)- 2- (3- (2,2,2- trifluoroacetyl group) benzyl) -9H- pyrimidos [4,5-b] indole -7-carboxylic acid's methyl esters (0.180g, 0.274mmol), the mixture of hydroxylamine hydrochloride (0.023g, 0.329mmol) and pyridine (0.355mL) in MeOH (1.9mL) adds Heat heats 48 hours to 65 DEG C.After reactant mixture to dry concentration, saturation NaHCO is utilized₃(15mL) washs residue Solution, in anhydrous MgSO₄Upper dry organic layer, filters and is concentrated to dryness, to provide the 4- ((3- as weak yellow foam ((3- ((tertbutyloxycarbonyl) amino) propyl group) (methyl) amino) propyl group) amino) -2- (3- (fluoro- 1- (the different nitrous of 2,2,2- tri- Base) ethyl) benzyl) -9H- pyrimidos [4,5-b] indole -7-carboxylic acid's methyl esters (184mg, 100% yield)：MS m/z 672.3(M +H)⁺；@254nm, the RT=2.01 minutes of 96.0%@220nm and of HPLC 91.4%.

4- ((3- ((3- ((tertbutyloxycarbonyl) amino) propyl group) are added portionwise in Ts-Cl (0.060g, 0.315mmol) (methyl) amino) propyl group) amino) -2- (3- (2,2,2- tri- fluoro- 1- (isonitroso) ethyl) benzyl) -9H- pyrimidos [4,5- B] indole -7-carboxylic acid's methyl esters (0.184g, 0.274mmol), DMAP (3.35mg, 0.027mmol) and triethylamine (0.048mL, 0.342mmol) in the mixture in DCM (12mL), to produce brown solution.After 1 hour, DCM (12mL) is utilized Diluted reaction mixture, washed using water (3 × 12mL), and in anhydrous MgSO₄Upper dry organic layer, filters and is concentrated to dryness It is dry, to provide 4- ((3- ((3- ((tertbutyloxycarbonyl) amino) propyl group) (methyl) amino) propyl group) ammonia as yellowish-brown foam Base) -2- (3- (tri- fluoro- 1- of 2,2,2- ((tosyl) imino group) ethyl) benzyl) -9H- pyrimidos [4,5-b] indoles -7- carboxylics Sour methyl esters (217mg, 96% yield)：MS m/z 826.2(M+H)⁺；The@254nm of 93.2%@220nm and of HPLC 91.3%, RT=2.20 minutes.

By 4- ((3- ((3- ((tertbutyloxycarbonyl) amino) propyl group) (methyl) amino) propyl group) amino) -2- (3- (2,2,2- Three fluoro- 1- ((tosyl) imino group) ethyls) benzyl) -9H- pyrimidos [4,5-b] indole -7-carboxylic acid's methyl esters (0.217g, 0.263mmol) solution in DCM (5.07mL) is cooled to -78 DEG C, and ammonia (1.7mL, 79mmol) is compressed into seal pipe In.Allow mixture to be slowly warmed to 20 DEG C, and stir 3 hours.After being cooled to -78 DEG C again, using with gas The pipe of the barrier film assembling sealing of outlet, and 20 DEG C are slowly warmed to, to evaporate ammonia.After 3 hours, mixture is concentrated To drying, then purified by fast chromatographic method, to provide 4- ((the 3- ((3- ((tertbutyloxycarbonyl) as white foam Amino) propyl group) (methyl) amino) propyl group) amino) -2- (the overlapping acridine -3- bases of 3- (3- trifluoromethyls)) benzyl) -9H- pyrimidines And [4,5-b] indole -7-carboxylic acid's methyl esters (139mg, 79% yield)：¹H NMR(400MHz,DMSO-d6)δppm 1.34(s, 9H) 1.52-1.62 (m, 2H) 1.72-1.89 (m, 2H) 2.08-2.25 (m, 3H) 2.30-2.44 (m, 4H) 2.94 (q, J= 6.4Hz, 2H) 3.64 (q, J=6.5Hz, 2H) 3.88 (s, 3H) 3.93 (d, J=8.4Hz, 1H) 4.04 (d, J=8.4Hz, 1H) 4.08 (s, 2H) 6.78 (br.s., 1H) 7.31-7.41 (m, 2H) 7.44-7.50 (m, 1H) 7.54 (t, J=5.5Hz, 1H) 7.59 (s, 1H) 7.83 (dd, J=8.4,1.4Hz, 1H) 7.99 (d, J=1.4Hz, 1H) 8.27 (d, J=8.4Hz, 1H) 12.07 (s, 1H)；MS m/z 671.4(M+H)⁺；The@220nm of HPLC 98.6% and 96.4%@254nm, RT=1.91 minutes.

Iodine (27.8mg, 0.110mmol) is added in 5mL round-bottomed flasks, is kept in dark place and is full of 4- ((3- ((3- in advance ((tertbutyloxycarbonyl) amino) propyl group) (methyl) amino) propyl group) amino) -2- (the overlapping acridine -3- bases of 3- (3- trifluoromethyls)) Benzyl) -9H- pyrimidos [4,5-b] indole -7-carboxylic acid's methyl esters (70mg, 0.104mmol) and triethylamine (43.6 μ L, 0.313mmol) the mixture in DCM (2mL), to produce yellow solution.After 20 DEG C are stirred 15 minutes, evaporation solvent Residue is purified with by fast chromatographic method, to produce 4- ((3- ((3- ((tertbutyloxycarbonyl) ammonia as weak yellow foam Base) propyl group) (methyl) amino) propyl group) amino) -2- (3- (3- trifluoromethyls) -3H- diazacyclo propane -3- bases) benzyl) - 9H- pyrimidos [4,5-b] indole -7-carboxylic acid's methyl esters (65mg, 0.097mmol, 93% yield)：¹H NMR(400MHz,DMSO- D6) δ ppm 1.33 (s, 9H) 1.53 (dt, J=13.8,7.0Hz, 2H) 1.70-1.82 (m, 2H) 2.15 (br.s., 3H) 2.24- 2.34(m,2H)2.34-2.42(m,2H)2.87-2.98(m,2H)3.55-3.66(m,2H)3.88(s,3H)4.10(s,2H) 6.76 (br.s., 1H) 7.14 (d, J=8.2Hz, 1H) 7.27 (s, 1H) 7.43 (t, J=7.8Hz, 1H) 7.49-7.58 (m, 2H) 7.83 (dd, J=8.2,1.4Hz, 1H) 7.99 (d, J=1.4Hz, 1H) 8.27 (d, J=8.2Hz, 1H) 12.06 (s, 1H)；MS m/z 669.2(M+H)⁺；The@220nm of HPLC 97.6% and 97.3%@254nm, RT=2.18 minutes.

Trifluoroacetic acid (0.400mL, 5.19mmol) is added to 4- ((3- ((3- ((tertbutyloxycarbonyl) amino) propyl group) (methyl) amino) propyl group) amino) -2- (3- (3- trifluoromethyls) -3H- diazacyclo propane -3- bases) benzyl) -9H- pyrimidos It is faint yellow molten to produce in solution of [4,5-b] the indole -7-carboxylic acid's methyl esters (0.064g, 0.096mmol) in DCM (4mL) Liquid.After 20 DEG C are stirred 30 minutes, using DCM (15mL) diluted reaction mixture, saturation NaHCO is utilized₃(10mL) is washed, With utilize DCM (10mL) aqueous layer extracted backward.In anhydrous MgSO₄The organic layer of upper dry combination, filters and is concentrated to dryness, with Intermediate 53C as weak yellow foam is provided：4- ((3- ((3- aminopropyls (methyl) amino) propyl group) amino) -2- (3- (3- Trifluoromethyl) -3H- diazacyclo propane -3- bases) benzyl) -9H- pyrimidos [4,5-b] indole -7-carboxylic acid's methyl esters (45mg, 83% yield)：HRMS m/z 569.2601(M+H)⁺；The@220nm of HPLC 97.1% and 96.9%@254nm, RT=1.96 point Clock.

By 2,5- dioxo pyrrolidin -1- bases -5- ((3aS, 4S, 6aR) -2- oxos hexahydro -1H- thiophene [3,4-d] miaows Azoles -4- bases) valerate (29.2mg, 0.085mmol) be added to 4- ((3- ((3- aminopropyls (methyl) amino) propyl group) amino) - 2- (3- (3- trifluoromethyls) -3H- diazacyclo propane -3- bases) benzyl) -9H- pyrimidos [4,5-b] indole -7-carboxylic acid's methyl esters In the mixture of (45mg, 0.079mmol) and triethylamine (16.55 μ L, 0.119mmol) in DMF (750 μ L), to produce Huang Color solution.After 20 DEG C are stirred 30 minutes, under high vacuum, reactant mixture is concentrated into light orange oil, and pass through Fast chromatographic method purifies residue, to produce 56mg white solid, from CH₃CN is lyophilized, solid as white to provide The compound of the embodiment 53 of body：4- ((3- (methyl (3- (5- ((3aS, 4S, 6aR) -2- oxygen hexahydro -1H- thiophene [3,4-d] miaows Azoles -4- bases) pentane amino) propyl group) amino) propyl group) amino) -2- (3- (3- trifluoromethyls) -3H- diazacyclo propane -3- bases) Benzyl) -9H- pyrimidos [4,5-b] indole -7-carboxylic acid's methyl esters (51mg, 0.064mmol, 81% yield)：¹H NMR(400MHz, DMSO-d₆)δppm 1.18-1.34(m,3H)1.35-1.50(m,3H)1.50-1.64(m,3H)1.70-1.82(m,2H)2.02 (t, J=7.4Hz, 2H) 2.15 (s, 3H) 2.26-2.34 (m, 2H) 2.37 (t, J=6.7Hz, 2H) 2.55 (d, J=12.5Hz, 1H) 2.77 (dd, J=12.1,5.1Hz, 1H) 2.99-3.10 (m, 3H) 3.56-3.66 (m, 2H) 3.88 (s, 3H) 4.03-4.14 (m, 1H) 4.10 (s, 2H) 4.26 (dd, J=7.6,5.3Hz, 1H) 6.34 (s, 1H) 6.39 (s, 1H) 7.14 (d, J=7.4Hz, 1H) 7.28 (s, 1H) 7.44 (t, J=7.8Hz, 1H) 7.52 (d, J=7.8Hz, 1H) 7.56 (t, J=5.3Hz, 1H) 7.73 (t, J=5.5Hz, 1H) 7.83 (dd, J=8.2,1.4Hz, 1H) 8.00 (d, J=1.4Hz, 1H) 8.28 (d, J=8.2Hz, 1H) 12.07(s,1H)；HRMS m/z 795.3368(M+H)⁺；The@220nm of HPLC 95.4% and 96.2%@254nm, RT=2.06 Minute.

Embodiment 55

By 2- benzyls -4- ((3- (piperidin-1-yl) propyl group) amino) -9H- pyrimidos [4,5-b] indoles in methylamine 2M - Solution of the 7- carboxylate methyl esters (embodiment 27,0.030g, 0.066mmol) in MeOH (10.00mL) is placed in the pipe of sealing, And 110 DEG C are heated to, heat 66 hours, then, mixture is cooled to 20 DEG C, is concentrated to dryness and passes through fast chromatographic Method purifies, and to provide 28mg colorless oil, is suspended in ether (2mL).After suspension caused by stirring 2 hours, Solid is collected on Buchner, block is washed using ether (2 × 0.5mL), and under high vacuum, production is dried at 40 DEG C Thing, until constant weight, to provide the embodiment 55 as white solid：Methyl 2- Benzyl-N-methyls -4- ((3- (piperidin-1-yl) Propyl group) amino) -9H- pyrimidos [4,5-b] indoles -7- formamides (23mg, 77% yield)：¹H NMR(400MHz,DMSO-d₆) δ ppm 1.38 (m, J=5.1Hz, 2H) 1.50 (five one group, J=5.5Hz, 4H) 1.80 (five one group, J=7.0Hz, 2H) 2.22-2.41 (m, 6H) 2.81 (d, J=4.3Hz, 3H) 3.58-3.68 (m, 2H) 4.03 (s, 2H) 7.15-7.21 (m, 1H) 7.27 (m, J=7.4,7.4Hz, 3H) 7.34-7.40 (m, 2H) 7.70 (dd, J=8.2,1.4Hz, 1H) 7.90 (d, J= 1.4Hz, 1H) 8.27 (d, J=8.2Hz, 1H) 8.46 (q, J=4.3Hz, 1H) 11.96 (s, 1H)；HRMS m/z 457.2708 (M+H)⁺；The@220nm of HPLC ＞ 99.5% and 98.9%@254nm, RT=1.53 minutes.

The HPLC retention times of report are used for reversed-phase HPLC (Agilent, 1200 series) solvent orange 2 A using following condition: MeOH∶H₂O∶TFA(5∶95∶0.05)；Solvent B:MeOH∶H₂O∶TFA(95∶5∶0.05)；Flow：3.0mL/min；In 2.0 minutes Gradient 0 is to 100%B；Post：ZorbaxC18,3.5 microns, 4.6x 30mm；Wavelength 220nm.

The structure of the embodiment of table 2., analytical HPLC retention times, LCMS data and biological data

EC50 is defined to, compared with medium culture medium (DMSO), causes CD34+CD45RA- cell counts 50% to increase Concentration.*EC₅₀: A ＞ 1000nM；B=500-1000nM；C=250-500nM；D=100-250；E=＜ 100nM；F=shows Show the compound of 1.3 times of amplifications of ＞.

In vitro functional analysis：

The ex vivo functional of the cell expanded using traditional culture CFU (CFU-C) experimental check.Passing Under conditions of system, it is inoculated into by untreated cell or using the cell of the compound incubation of DMSO, positive control or the present invention In methylcellulose medium.As example, compound 1 (table 2, embodiment 1) expands many pluripotency HPCs.Utilize The methylcellulose culture of the 1000CD34+mPB cells of the processing of compound 1 causes multidirectional granulocyte red blood cell, macrophage thin for 10 days Born of the same parents and megacaryocyte (GEMM clones), which exceed 5- times of cell of input, to be increased, and compared with control cell, 10 times of increases.This shows Compound 1 promotes the amplification of pluripotent progenitor cells.

In vivo functionality is analyzed：

Lacked using the CD34+mPB cell grafting immune deficiency bacterial strain NOD severe combined immunizations of the compound culture of the present invention Fall into gamma (immunodeficient strain NOD scid gamma) (NSG) mouse.Compound 1 will be utilized (table 2, to implement Example 1) or vehicle control condition be incubated the achievement of 2,000,000 and 500,000CD34+mPB cells of 10 days and be transplanted to NSG mouse In.After the transfer after 8 weeks, rebuild using the human hematopoietic cell in the antibody test NSG marrow to people CD45.Utilize compound 1 rather than the cell of the processing that utilizes the medium being capable of grafting NSG mouse.In addition, the reconstruction of human marrow and lymph compartment is also confirmed that, Because bone marrow cell is respectively positive to the mankind CD33+ and CD19+.These results are shown, utilize the CD34 of the amplification of compound 1 + mPB not only facilitates multidirectional population in grafting and retention body and recovers potential.

The combination of compound：

Lacked using the CD34+mPB cell grafting immune deficiency bacterial strain NOD severe combined immunizations of the compound culture of the present invention Fall into gamma (NSG) mouse.2,000,000 He of compound 1 (table 2, embodiment 1) or the incubation 10 days of vehicle control condition will be utilized The achievement of 500,000CD34+mPB cells is transplanted in NSG mouse.After the transfer after 8 weeks, the antibody inspection to people CD45 is utilized The human hematopoietic cell tested in NSG marrow is rebuild.Using compound 1 rather than the cell for the processing that utilizes the medium can grafting NSG it is old Mouse.In addition, the reconstruction of human marrow and lymph compartment is also confirmed that, because bone marrow cell is sun to the mankind CD33+ and CD19+ respectively Property.These results indicate that the CD34+mPB expanded using compound 1 not only facilitates multidirectional population in grafting and retention body Recover potential.

It should be understood that the purpose that embodiment described herein and embodiment are merely to illustrate, and this area will be suggested Technical staff carry out according to its various changes or change and they be included within the present disclosure appearance and appended claims In the range of.

Claims

A kind of 1. compounds of formula II

Or its salt,

Wherein：

Z is-C (O) OMe ,-C (O) OEt, CN,Or-C (O) NHMe

W is

R²It is-H ,-Me,

Condition is that the compounds of formula II is not
A kind of 2. Formula II A compound

Or its salt,

Wherein R¹It is Me or Et and W and R²Individually according to defined in claim 1.
A kind of 3. formula IIB compound

Or its salt,

Wherein W and R²Individually according to defined in claim 1, and Het is
4. compound according to any one of claim 1 to 3, wherein, R²It is benzyl.
5. compound according to claim 1, wherein：

Z is CO₂Me or 2- methyl -2H- tetrazolium -5- bases；

R²It is benzyl or H.
6. compound according to claim 5, wherein, R²It is H.
7. compound according to claim 5, wherein, W is
8. compound according to claim 7, wherein, Z is
9. compound according to claim 8, wherein, R²It is H.
10. a kind of compound, the compound are：

Or compound 1-18 salt, compound 21-25 salt, compound 33-34 salt, compound 37-40 salt, compound 42-48 salt and compound 50-55 salt.
11. a kind of compound, the compound are：
12. a kind of compound, the compound are：

Or its salt.
A kind of 13. medicine of the compound or its salt and pharmaceutical carrier defined in any one including according to claim 1 to 12 Compositions.
14. prepared including the reagent according to the compound or its salt defined in any one of claim 1 to 12 for increasing Add the application in the medicine of stem cell and/or progenitor cells, wherein external or initial cell population is connect with the reagent in vitro Touch.
15. application according to claim 14, the application is in vitro.
16. the application according to claims 14 or 15, including the initial cell population is expanded with least one cell The factor contacts.
17. the application according to claims 14 or 15, wherein, initial cell population include the peripheral blood (mPB) from mobilization, The CD34+ cells of marrow (BM) or Cord blood (UCB) harvest.
18. compound or its salt defined at least one any one according to claim 1 to 12 is alternatively with least one The kind cell proliferation factor application in the medicine for amplifying candidate stem cell is prepared together, wherein at least one describedization In the presence of compound or its salt, initial cell population is cultivated in vitro or in vitro.
19. pharmaceutical composition according to claim 13, described pharmaceutical composition is suitable for being injected intravenously.
It is used to increasing the kits of stem cell and/or progenitor cells 20. a kind of, including in any one according to claim 1 to 12 The compound or its salt and operation instructions limited.
21. a kind of kit for amplifying candidate stem cell, including according to defined in any one of claim 1 to 12 Compound or its salt and operation instructions, alternatively, the kit include at least one cell proliferation factor.
22. compound according to claim 12, wherein, the salt of the compound is Hydrobromate.
23. the application according to claim 14 or 18, wherein, the initial cell population is by one or two Cord blood list The CD34+ cells composition of member purifying.
24. application according to claim 23, wherein, in the presence of the compound, the initial cell population is trained Support 2 days to 21 days.
25. the application according to claim 14 or 18, it is dense between 1nM and 3000nM to give the initial cell population The compound of degree.
26. the application according to claim 16 or 18, wherein, the cell proliferation factor is interleukin 3 (IL- 3), Granulocyte Colony-stimulating (GM-CSF), thrombopoietin (TPO), FLT3L (FLT3-L), stem cell factor (SCF), interleukin-6 (IL-6) or combinations thereof.
27. application according to claim 26, wherein, the cell proliferation factor be SCF, FLT3-L, TPO, IL-6 or Combinations thereof.
28. application according to claim 18, wherein, at least one cell proliferation factor is StemRegenin 1 (SR1)。